RNAi modulation of APOB and uses thereof

ABSTRACT

The invention relates to compositions and methods for modulating the expression of apolipoprotein B, and more particularly to the downregulation of apolipoprotein B by chemically modified oligonucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/728,139, filed Mar. 19, 2010, which is a divisionalapplication of U.S. patent application Ser. No. 12/400,744, filed Mar.9, 2009, now issued as U.S. Pat. No. 7,723,317, which is a divisionalapplication of U.S. patent application Ser. No. 11/235,385, filed Sep.26, 2005, now issued as U.S. Pat. No. 7,528,118, and which claimspriority to U.S. Provisional Application Ser. No. 60/613,141, filed Sep.24, 2004; each of which is incorporated herein by reference in itsentirety.

SEQUENCE LISTING

In accordance with 37 C.F.R. §1.821(c), this application is filed withan electronically submitted Sequence Listing in ASCII text format (68kb), entitled “16562_US_Sequence_Listing,” created on Mar. 19, 2010.This Sequence Listing is hereby incorporated by reference, in itsentirety and for all purposes.

TECHNICAL FIELD

The invention relates to compositions and methods for modulating theexpression of apolipoprotein B, and more particularly to thedownregulation of apolipoprotein B by oligonucleotides, e.g., chemicallymodified oligonucleotides.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.,Nature 391:806-811, 1998). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function.

Lipoproteins consist of acylglycerols and cholesteryl esters surroundedby an amphiphilic coating of protein, phospholipid and cholesterol. Theprotein components of lipoproteins are known as apolipoproteins, and atleast nine apolipoproteins exist in humans. Apolipoprotein B (ApoB) isfound in various classes of lipoproteins: chylomicrons, very low densitylipoproteins (VLDL), intermittent density lipoproteins (IDL), and lowdensity lipoproteins (LDL). ApoB functions as a recognition signal forthe cellular binding and internalization of LDL particles by the ApoB/Ereceptor. An accumulation or overabundance of apolipoproteinB-containing lipoproteins can lead to lipid-related disorders such asatherosclerosis.

The development of therapies that reduce ApoB can be useful for treatinglipid-related disorders. One oligonucleotide based therapy, in the formof antisense therapy, has been shown to reduce ApoB levels in mouse invivo, and treatments subsequently reduced serum cholesterol andtriglyceride levels (U.S. Publication No. 2003/0215943). These resultsdemonstrated a moderate downregulation of ApoB and its use as a targetin treating lipid-related disorders. The present invention advances theart by providing IRNA agents that have been shown to reduce serum ApoBlevels in vivo.

SUMMARY

The invention provides compositions and methods for reducingapolipoprotein B (ApoB) levels in a subject, e.g., a mammal, such as ahuman. The method includes administering to a subject an iRNA agent thatsilences an ApoB gene. The iRNA agent can be one described here, or canbe a dsRNA that is based on one of the active sequences and target anidentical region of the ApoB gene, e.g., a mammalian ApoB gene, such asan ApoB gene from a human or mouse. The iRNA agent can comprise lessthan 30 nucleotides per strand, e.g., 21-23 nucleotides and consist of,comprise or be derived from one of the agents provided herein underagent numbers 1-74. These preferred iRNA agents include four or morenucleotide mismatches to all non-ApoB gene sequences in the subject.

The invention specifically provides an iRNA agent that includes a sensestrand having at least 15 contiguous nucleotides of the sense strandsequences, and an antisense strand having at least 15 contiguousnucleotides of the antisense sequences, of the iRNA agents providedherein under agent numbers 1-74, e.g. agent number 1, sense strandsequence 5′-cuuuacaagccuugguucagu-3′ (SEQ. ID NO. 153), antisense strandsequence 5′-acugaaccaaggcuuguaaagug-3′(SEQ. ID No. 154).

It shall be understood that, while some of the iRNA agents providedherein encompass specific preferred patterns of modified nucleotides,e.g. agent numbers 54 to 74, the iRNA agents of agent numbers 1-53 areprovided as blueprints. They are meant to encompass such modificationsas are evident to the skilled person as equivalent to the iRNA agents ofagent numbers 1-53, such as are further described below, e.g.2′-O-methyl modifications, generic base substitutions, etc, that theskilled person would not expect to alter the properties of these agents,and specifically the ability of the two strands to hybridize understringent conditions with their complementary counterparts.

TABLE 1 Exemplary iRNA agents to target ApoB SEQ. SEQ. ID ID DuplexAgent No. Sequence sense strand^(a) No. Sequence antisense strand^(a)descriptora number 153

uuuacaagccuugguucagu 154

cugaaccaaggcuuguaaagug AL-DUP 5097 1 155

gaaucuuauauuugauccaa 156

uggaucaaauauaagauucccu AL-DUP 5098 2 161

agaagggaaucuuauauuug 162

aaauauaagauucccuucuauu AL-DUP 5101 3 147

ccccaucacuuuacaagccu 148

ggcuuguaaagugauggggcug AL-DUP 5094 4 159

aauagaagggaaucuuauau 160

uauaagauucccuucuauuuug AL-DUP 5100 5 145

cacauccuccaguggcugaa 146

ucagccacuggaggaugugagu AL-DUP 5093 6 49

gguguauggcuucaacccug 50

aggguugaagccauacaccucu AL-DUP 5024 7 127

ugaacaucaagaggggcauc 128

augccccucuugauguucagga AL-DUP 5084 8 137

aguuugugacaaauaugggc 138

cccauauuugucacaaacucca AL-DUP 5089 9 93

ucaagugucaucacacugaa 94

ucagugugaugacacuugauuu AL-DUP 5046 10 97

ucaucacacugaauaccaau 98

uugguauucagugugaugacac AL-DUP 5048 11 95

caagugucaucacacugaau 96

uucagugugaugacacuugauu AL-DUP 5047 12 99

uguccauucaaaacuaccac 100

ugguaguuuugaauggacaggu AL-DUP 5049 13 129

gaacaucaagaggggcauca 130

gaugccccucuugauguucagg AL-DUP 5085 14 135

gccccaucacuuuacaagcc 136

gcuuguaaagugauggggcugg AL-DUP 5088 15 131

uccagccccaucacuuuaca 132

guaaagugauggggcuggacac AL-DUP 5086 16 27

guguauggcuucaacccuga 28

caggguugaagccauacaccuc AL-DUP 5013 17 107

accuguccauucaaaacuac 108

uaguuuugaauggacaggucaa AL-DUP 5053 18 143

uugggaagaagaggcagcuu 144

agcugccucuucuucccaauua AL-DUP 5092 19 123

aacacuaagaaccagaagau 124

ucuucugguucuuaguguuagc AL-DUP 5061 20 57

agguguauggcuucaacccu 58

ggguugaagccauacaccucuu AL-DUP 5028 21 41

uguauggcuucaacccugag 42

ucaggguugaagccauacaccu AL-DUP 5020 22 157

aagggaaucuuauauuugau 158

ucaaauauaagauucccuucua AL-DUP 5099 23 59

ggcuucaacccugagggcaa 60

ugcccucaggguugaagccaua AL-DUP 5029 24 63

uauggcuucaacccugaggg 64

ccucaggguugaagccauacac AL-DUP 5031 25 121

aagugucaucacacugaaua 122

auucagugugaugacacuugau AL-DUP 5060 26 55

aaaucaagugucaucacacu 56

gugugaugacacuugauuuaaa AL-DUP 5027 27 39

ugacaaauaugggcaucauc 40

augaugcccauauuugucacaa AL-DUP 5019 28 71

accaacuucuuccacgaguc 72

acucguggaagaaguugguguu AL-DUP 5035 29 73

augaacaccaacuucuucca 74

ggaagaaguugguguucaucug AL-DUP 5036 30 105

ccuguccauucaaaacuacc 106

guaguuuugaauggacagguca AL-DUP 5052 31 61

aacaccaacuucuuccacga 62

cguggaagaaguugguguucau AL-DUP 5030 32 45

auaccguguauggaaacugc 46

caguuuccauacacgguaucca AL-DUP 5022 33 133

agccccaucacuuuacaagc 134

cuuguaaagugauggggcugga AL-DUP 5087 34 5

auugauugaccuguccauuc 6

aauggacaggucaaucaaucuu AL-DUP 5002 35 77

gaugaacaccaacuucuucc 78

gaagaaguugguguucaucugg AL-DUP 5038 36 1

agccuugguucaguguggac 2

uccacacugaaccaaggcuuga AL-DUP 5000 37 117

caucacacugaauaccaaug 118

auugguauucagugugaugaca AL-DUP 5058 38 3

gaacaccaacuucuuccacg 4

guggaagaaguugguguucauc AL-DUP 5001 39 69

caccaacuucuuccacgagu 70

cucguggaagaaguugguguuc AL-DUP 5034 40 25

gauugaccuguccauucaaa 26

uugaauggacaggucaaucaau AL-DUP 5012 41 21

aaauggacucaucugcuaca 22

guagcagaugaguccauuugga AL-DUP 5010 42 29

cugugggauuccaucugcca 30

ggcagauggaaucccacagacu AL-DUP 5014 43 109

aucacacugaauaccaaugc 110

cauugguauucagugugaugac AL-DUP 5054 44 23

auugaccuguccauucaaaa 24

uuugaauggacaggucaaucaa AL-DUP 5011 45 33

caauuugaucaguauauuaa 34

uaauauacugaucaaauuguau AL-DUP 5016 46 83

caagccuugguucagugugg 84

cacacugaaccaaggcuuguaa AL-DUP 5041 47 79

uuccaucugccaucucgaga 80

cucgagauggcagauggaaucc AL-DUP 5039 48 43

accguguauggaaacugcuc 44

agcaguuuccauacacgguauc AL-DUP 5021 49 35

gacucaucugcuacagcuua 36

aagcuguagcagaugaguccau AL-DUP 5017 50 51

uuugugacaaauaugggcau 52

ugcccauauuugucacaaacuc AL-DUP 5025 51 65

uggcuucaacccugagggca 66

gcccucaggguugaagccauac AL-DUP 5032 52 125

aauuugaucaguauauuaaa 126

uuaauauacugaucaaauugua AL-DUP 5062 53 ^(a)See Table 2 for an explanationof nucleotide representation (e.g., lower case letters, bold anditalicized letters).

As shown in Example 3 hereinbelow, the iRNA agents of Table 1, agentnumbers 1-53, possess the advantageous and surprising ability to reducethe amount of ApoB mRNA present in cultured human HepG2 cells afterincubation with these iRNA agents by more than 50% compared to cellswhich have not been incubated with the iRNA agent, and/or to reduce theamount of ApoB protein secreted into cell culture supernatant bycultured human HepG2 cells by more than 50% (see Table 8).

The invention further provides an iRNA agent that includes a sensestrand having at least 15 contiguous nucleotides of the sense sequencesof the iRNA agents, agent numbers 1-19, 24-26, 29, 30 and 32-42, and anantisense strand having at least 15 contiguous nucleotides of theantisense sequences of the iRNA agents, agent numbers 1-19, 24-26, 29,30 and 32-42. As shown in Example 3 hereinbelow, the iRNA agents, agentnumbers 1-19, 24-26, 29, 30 and 32-42, possess the advantageous andsurprising ability to reduce the amount of ApoB mRNA present in culturedhuman HepG2 cells after incubation with these iRNA agents by more than60% compared to cells which have not been incubated with the iRNA agent,and/or to reduce the amount of ApoB protein secreted into cell culturesupernatant by more than 60% (see Table 8).

The invention further provides an iRNA agent that includes a sensestrand having at least 15 contiguous nucleotides of the sense sequencesof the agents provided in Table 1, agent numbers 1-12, 15, 17, 24, 29,30 and 32-35, and an antisense strand having at least 15 contiguousnucleotides of the antisense sequences of the agents provided in Table1, agent numbers 1-12, 15, 17, 24, 29, 30 and 32-35. As shown in Example3 hereinbelow, these iRNA agents possess the advantageous and surprisingability to reduce the amount of ApoB mRNA present in cultured humanHepG2 cells after incubation with these agents by more than 70% comparedto cells which have not been incubated with the agent, and/or to reducethe amount of ApoB protein secreted into cell culture supernatant bymore than 70% (see Table 8).

The invention further provides an iRNA agent that includes a sensestrand having at least 15 contiguous nucleotides of the sense sequencesof the iRNA agents, agent numbers 1-5, 7, and 11, and an antisensestrand having at least 15 contiguous nucleotides of the antisensesequences of the iRNA agents, agent numbers 1-5, 7, and 11. As shown inExample 3 hereinbelow, these iRNA agents possess the advantageous andsurprising ability to reduce the amount of ApoB mRNA present in culturedhuman HepG2 cells after incubation with these agents by more than 80%compared to cells which have not been incubated with the agent, and/orto reduce the amount of ApoB protein secreted into cell culturesupernatant by more than 80% (see Table 8).

In a particularly preferred aspect, the iRNA agent is selected from thegroup of: the iRNA agent, agent number 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74.

In another preferred embodiment, the iRNA agent reduces the amount ofApoB mRNA present in cultured human HepG2 cells after incubation withthe iRNA agent by more than 50% compared to cells which have not beenincubated with the agent, and/or reduces the amount of ApoB proteinsecreted into cell culture supernatant by cultured human HepG2 cells bymore than 50%, and/or reduces the amount of apo-B mRNA present in murineliver cells of C57Bl/6 mice by at least 20% in vivo after administrationof 50 mg/kg body weight or 100 mg/kg body weight.

Further provided by the instant invention are iRNA agents comprising asense strand and antisense strand each comprising a sequence of at least16, 17 or 18 nucleotides which is essentially identical, as definedbelow, to one of the sequences of the iRNA agents, agent numbers 1-74,except that not more than 1, 2 or 3 nucleotides per strand,respectively, have been substituted by other nucleotides (e.g. adenosinereplaced by uracil), while essentially retaining the ability to inhibitApoB expression in cultured human HepG2 cells, as defined below.

In one embodiment, the iRNA agent is at least 15 nucleotides long andincludes a sense RNA strand and an antisense RNA strand, wherein theantisense RNA strand is 30 or fewer nucleotides in length, and theduplex region of the iRNA agent is 15-30, preferably 18-25 nucleotidepairs in length. The iRNA agent may further include a nucleotideoverhang having 1 to 4, preferably 2 to 3, unpaired nucleotides, and theunpaired nucleotides may have at least one phosphorothioate dinucleotidelinkage. The nucleotide overhang can be, e.g., at the 3′-end of theantisense strand of the iRNA agent.

In one embodiment, the iRNA agent inhibits the expression of human andmouse ApoB, e.g. in human HepG2 and mouse NmuLi cells.

In one embodiment, and as described herein, it is preferred that theIRNA agent be modified by attachment of a hydrophobic moiety, e.g. acholesterol-comprising moiety, preferably to the sense strand of theiRNA agent, and more preferably to the 3′-end of the sense strand of theiRNA agent.

In another embodiment, and as described herein, it is preferred that theiRNA agent be modified to improve stability. Preferred modifications arethe introduction of phosphorothioate linkages and 2′-substitutions onthe ribose unit, e.g., 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA) substitutions.

Preferably, these 2′-substitutions are made to the 5′ nucleotide of a5′-UA-3′ dinucleotide, a 5′-UG-3′ dinucleotide, a 5′-CA-3′ dinucleotide,a 5′-UU-3′ dinucleotide, or a 5′-CC-3′ dinucleotide on the sense strandand, optionally, also on the antisense strand of the iRNA agent, or toall pyrimidine-base comprising nucleotides. More, preferably, the5′-most pyrimidines in all occurrences of the sequence motifs 5′-UA-3′,5′-CA-3′, 5′-UU-3′, and 5′-UG-3′ are 2′-modified nucleotides. Yet morepreferably, all pyrimidines in the sense strand are 2′-modifiednucleotides, and the 5′-most pyrimidines in all occurrences of thesequence motifs 5′-UA-3′ and 5′-CA-3′. Most preferably, all pyrimidinesin the sense strand are 2′-modified nucleotides, and the 5′-mostpyrimidines in all occurrences of the sequence motifs 5′-UA-3′,5′-CA-3′, 5′-UU-3′, and 5′-UG-3′ are 2′-modified nucleotides in theantisense strand.

In another embodiment, and as described herein, a cholesterol moiety(e.g., on the 3′-end of the sense strand), a 2′-modification (e.g., a2′-O-methyl or 2′-deoxy-2′-fluoro-modification), and a phosphorothioate(e.g., on the 3′-most one or two nucleotides of the sense and antisensestrands) are present in the same iRNA agent.

In a preferred embodiment, administration of an iRNA agent, e.g., aniRNA agent described herein, is for treatment of a disease or disorderpresent in the subject in which ApoB expression plays a role. In anotherpreferred embodiment, administration of the iRNA agent is forprophylactic treatment of ApoB mediated disorders.

In one aspect, the invention features preparations, includingsubstantially pure or pharmaceutically acceptable preparations of iRNAagents which modulate e.g., inhibit, ApoB. The preparations can includean iRNA agent that targets an ApoB encoding nucleic acid and apharmaceutically acceptable carrier. In one embodiment, the iRNA agenthas a sense strand having at least 15 contiguous nucleotides of thesense sequences of the iRNA agents, agent numbers 1-74, and an antisensestrand having at least 15 contiguous nucleotides of the antisensesequences of the iRNA agents, agent numbers 1-74.

In another aspect, the invention features a method of preparing apharmaceutical composition, comprising formulating an iRNA agent a sensestrand having at least 15 contiguous nucleotides of the sense sequencesof the iRNA agents, agent numbers 1-74, and an antisense strand havingat least 15 contiguous nucleotides of the antisense sequences of theiRNA agents, agent numbers 1-74, with a pharmaceutically acceptablecarrier.

The pharmaceutical composition of the invention can be administered inan amount sufficient to reduce expression of ApoB messenger RNA (mRNA).In one embodiment, the iRNA agent is administered in an amountsufficient to reduce expression of ApoB protein (e.g., by at least 2%,4%, 6%, 10%, 15%, 20% or greater).

The pharmaceutical composition of the invention can be administered to asubject, wherein the subject is at risk for or suffering from a disordercharacterized by elevated or otherwise unwanted expression of ApoB,elevated or otherwise unwanted levels of cholesterol, a lipid-mediatedvascular disorder, and/or disregulation of lipid metabolism. The iRNAagent can be administered to an individual diagnosed with or having thedisorder, or at risk for the disorder to delay onset of the disorder ora symptom of the disorder. These disorders include HDL/LDL cholesterolimbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL),acquired hyperlipidemia; hypercholestorolemia; statin-resistanthypercholesterolemia; coronary artery disease (CAD); coronary heartdisease (CHD); thrombosis; and atherosclerosis. In one embodiment, theiRNA that targets ApoB is administered to a subject suffering fromstatin-resistant hypercholesterolemia.

The pharmaceutical composition of the invention can be administered inan amount sufficient to reduce levels of serum LDL cholesterol and/orHDL cholesterol and/or total cholesterol in a subject. For example, theiRNA is administered in an amount sufficient to decrease totalcholesterol by at least 0.5%, 1%, 2.5%, 5%, 10% or more in the subject.In one embodiment, the pharmaceutical composition of the invention isadministered in an amount sufficient to reduce the risk of myocardialinfarction in the subject. In a preferred embodiment the pharmaceuticalcomposition is administered repeatedly.

In one embodiment, the iRNA agent can be targeted to the liver, and ApoBexpression levels are decreased in the liver following administration ofthe ApoB iRNA agent. For example, the iRNA agent can be complexed with amoiety that targets the liver, e.g., an antibody or ligand, such ascholesterol that binds a receptor on liver cells. As shown in Example7G) below, the conjugation of a cholesterol-comprising moiety led toefficient uptake of siRNAs by liver tissue and decreased ApoB mRNAlevels in liver samples. This shows that modifications such as aconjugation with a cholesterol-comprising moiety allows for the use ofiRNA agents in vivo to target genes in the liver.

In one embodiment, the iRNA agent can be targeted to the gut, e.g., tothe intestine, such as to the jejunum of the intestine, and ApoBexpression levels are decreased in the gut following administration ofthe ApoB iRNA agent. Unexpectedly, it was found that an iRNA agentconjugated to a cholesterol moiety can be used to target an IRNA agentto the gut. As shown in Example 7G) below, the conjugation of acholesterol-comprising moiety led to efficient uptake of siRNAs byintestinal tissues and decreased ApoB mRNA levels in intestinal tissuesamples. This shows that modifications such as a conjugation with acholesterol-comprising moiety allows for the use of iRNA agents in vivoto target genes in tissues of the gut.

In one embodiment, the iRNA agent has been modified, or is associatedwith a delivery agent, e.g., a delivery agent described herein, e.g., aliposome. In one embodiment, the modification mediates association witha serum albumin (SA), e.g., a human serum albumin (HSA), or a fragmentthereof.

A method of evaluating an iRNA agent thought to inhibit the expressionof an ApoB-gene, the method comprising:

-   -   a. providing an iRNA agent, wherein a first strand is        sufficiently complementary to a nucleotide sequence of an ApoB        mRNA, and a second strand is sufficiently complementary to the        first strand to hybridize to the first strand;    -   b. contacting the iRNA agent to a cell comprising an ApoB gene;    -   c. comparing ApoB gene expression before contacting the iRNA        agent to the cell, or of uncontacted control cells, to the ApoB        gene expression after contacting the iRNA agent to the cell; and    -   d. determining whether the iRNA agent is useful for inhibiting        ApoB gene expression, wherein the iRNA is useful if the amount        of ApoB RNA present in the cell, or protein secreted by the        cell, is less than the amount prior to contacting the iRNA agent        to the cell.

In one embodiment, steps b.-d. are performed both in vitro and innon-human laboratory animals in vivo. In another embodiment. The methodfurther comprises determining the activity of the iRNA agent inactivating interferon-α production by peripheral blood mononuclearcells.

The methods and compositions of the invention, e.g., the methods andcompositions to treat diseases and disorders of the liver describedherein, can be used with any of the iRNA agents described. In addition,the methods and compositions of the invention can be used for thetreatment of any disease or disorder described herein, and for thetreatment of any subject, e.g., any animal, any mammal, such as anyhuman.

The methods and compositions of the invention, e.g., the methods andiRNA compositions to treat lipid metabolism disorders described herein,can be used with any dosage and/or formulation described herein, as wellas with any route of administration described herein.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thisdescription, the drawings, and from the claims. This applicationincorporates all cited references, patents, and patent applications byreferences in their entirety for all purposes.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic illustrating the synthesis and structure ofcholesterol conjugated RNA strands. The sphere represents the solidphase (controlled pore glass, CPG).

FIG. 2 is a graph depicting the ratio [ApoB mRNA]/[GAPDH control mRNA]following treatment of cells with increasing levels of siRNA,AL-DUP5024. Determination of the inhibitor concentration at 50% maximalinhibition (IC₅₀) was determined by curve fitting using the computersoftware Xlfit using the following parameters: Dose Response One Site, 4Parameter Logistic Model, fit=(A+((B−A)/(1+(((10^C)/x)^D)))),inv=((10^C)/((((B−A)/(y−A))−1)^(1/D))), res=(y−fit).

FIG. 3 is a panel of polyacrylamide gels depicting the degradation ofsiRNA duplexes AL-DUP 5024, AL-DUP 5163, AL-DUP 5164, AL-DUP 5165,AL-DUP 5166, AL-DUP 5180, and AL-DUP 5181 by mouse serum nucleases.siRNA duplexes were incubated in mouse serum for 0, 1, 3, 6 or 24 hours.The lanes marked “unb” represent an untreated control.

FIG. 4 is a panel of polyacrylamide gels depicting the degradation ofsiRNA duplexes AL-DUP 5167, AL-DUP 5168, AL-DUP 5048, AL-DUP 5169,AL-DUP 5170, AL-DUP 5182, and AL-DUP 5183 by mouse serum nucleases.siRNA duplexes were incubated in mouse serum for 0, 1, 3, 6 or 24 hours.The lanes marked “unb” represent an untreated control.

FIG. 5A is a dose-response plot of ApoB protein secretion intosupernatant by cultured human HepG2 cells incubated with mediacontaining 100, 33, 11, 3.7, 1.2, 0.4, 0.14, or 0.05 nM of ApoB-specificsiRNA duplex AL-DUP 5163. The response is expressed as the ratio of ApoBprotein concentrations in the supernatant of cells treated with theApoB-specific siRNA duplex to the ApoB concentration in the supernatantof cells treated with an unspecific control siRNA duplex with (AL-DUP5129, diamonds) or without (AL-DUP HCV, squares)cholesterol-conjugation.

FIG. 5B is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5164 at the concentration ranges described for FIG. 5A.

FIG. 5C is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5165 at the concentration ranges described for FIG. 5A.

FIG. 5D is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5166 at the concentration ranges described for FIG. 5A.

FIG. 5E is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5180 at the concentration ranges described for FIG. 5A.

FIG. 5F is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5181 at the concentration ranges described for FIG. 5A.

FIG. 5G is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5167 at the concentration ranges described for FIG. 5A.

FIG. 5H is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5168 at the concentration ranges described for FIG. 5A.

FIG. 5I is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5169 at the concentration ranges described for FIG. 5A.

FIG. 5J is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5170 at the concentration ranges described for FIG. 5A.

FIG. 5K is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5182 at the concentration ranges described for FIG. 5A.

FIG. 5L is a dose-response plot of ApoB protein secretion according tothe method described in FIG. 5A. The HepG2 cells were incubated with thesiRNA duplex 5183 at the concentration ranges described for FIG. 5A.

FIG. 6A to D depict, by way of example, results obtained in experimentsdescribed in Example 7, (H), below.

FIG. 6A is an S1-nuclease protection assay with radiolabeled probescomplementary to antisense strands of siRNAs. The assay was used todetect siRNAs in pooled liver and jejunum tissue lysates from animalsinjected with saline (“−”), AL-DUP 5386 (“A”), AL-DUP 5311 (“B”), AL-DUP5385 (“C2”), and AL-DUP 5167 (“C1”). The three cholesterol-conjugatedsiRNAs were detected at comparable levels in liver and jejunum, but thenon-cholesterol-conjugated siRNA AL-DUP 5385 remained below detectionlevels in both tissues. S1-nuclease protection assay for endogeneousmiRNAs served as a loading controls for jejunum (miRNA 143, sequence5′-UGAGAUGAAGCACUGUAGCUCA-3′, SEQ. ID NO. 270) and liver (miRNA 122,sequence 5′-UGGAGUGUGACAAUGGUGUUUG-3′, SEQ. ID NO. 269).

FIG. 6B is a graph depicting the results of branched-DNA assays todetect ApoB mRNA levels in mouse liver and jejunum tissue followingsiRNA treatment. Tissue lysates were used for ApoB and GAPDH mRNAquantification and the ratio of ApoB and GAPDH mRNA was calculated andexpressed as a group average relative to a saline control group. Barsrepresent group mean values. Error bars represent the standard deviationof the mean. Asterisks above bars in bar graphs denote groupssignificantly different compared to saline control animals at p<0.01.

FIG. 6C is a graph depicting the results of ELISA assays to measureplasma ApoB protein levels following siRNA treatment. ApoB-100 fromplasma samples of individual animals was detected using the primaryantibody LF3 against mouse ApoB-100. Mean group values of ApoB proteinlevel are represented relative to the mean of saline control. Barsrepresent group mean values. Error bars represent the standard deviationof the mean. Asterisks above bars in bar graphs denote groupssignificantly different compared to saline control animals p<0.01.

FIG. 6D is a graph depicting total plasma ApoB protein levels followingsiRNA treatment. Total plasma cholesterol levels where measured usingthe Cholesterol detection kit (Diasys). Bars represent group meanvalues. Error bars represent the standard deviation of the mean.Asterisks above bars in bar graphs denote groups significantly differentcompared to saline control animals p<0.01.

FIG. 7A is a schematic representation of the ApoB mRNA and of theadapter ligated ApoB cDNA used for 5′-RACE PCR. The schematic shows therelative target sites of the AL-DUP 5167 siRNA and the PCR primers, andthe size of PCR reaction products.

FIG. 7B is an agarose gel of RACE-PCR amplification 3. Theelectrophoretic analysis indicates specific cleavage products in liverand jejunum of mice treated with ApoB specific AL-DUP 5167 only. Thelanes of the gel are marked by capital letters that indicate treatmentgroups and controls. The lanes are marked as follows: A: PBS; B: AL-DUP5386; C: AL-DUP 5167; D: AL-DUP 5163, E: AL DUP 5385; F: AL-DUP 5311;Fc: Control, Forward primer only using cDNA from group C; RC: Control,Reverse primer only using cDNA from group C.

DETAILED DESCRIPTION

For ease of exposition the term “nucleotide” or “ribonucleotide” issometimes used herein in reference to one or more monomeric subunits ofan RNA agent. It will be understood that the usage of the term“ribonucleotide” or “nucleotide” herein can, in the case of a modifiedRNA or nucleotide surrogate, also refer to a modified nucleotide, orsurrogate replacement moiety, as further described below, at one or morepositions.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are described herein. While numerousmodified RNAs and nucleoside surrogates are described, preferredexamples include those which have greater resistance to nucleasedegradation than do unmodified RNAs. Preferred examples include thosethat have a 2′ sugar modification, a modification in a single strandoverhang, preferably a 3′ single strand overhang, or, particularly ifsingle stranded, a 5′-modification which includes one or more phosphategroups or one or more analogs of a phosphate group.

An “iRNA agent” (abbreviation for “interfering RNA agent”) as usedherein, is an RNA agent, which can downregulate the expression of atarget gene, e.g., ApoB. While not wishing to be bound by theory, aniRNA agent may act by one or more of a number of mechanisms, includingpost-transcriptional cleavage of a target mRNA sometimes referred to inthe art as RNAi, or pre-transcriptional or pre-translational mechanisms.An iRNA agent can include a single strand or can include more than onestrands, e.g., it can be a double stranded (ds) iRNA agent. If the iRNAagent is a single strand it is particularly preferred that it include a5′ modification which includes one or more phosphate groups or one ormore analogs of a phosphate group.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpanhandle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule.

A “ds iRNA agent” (abbreviation for “double stranded iRNA agent”). asused herein, is an iRNA agent which includes more than one, andpreferably two, strands in which interchain hybridization can form aregion of duplex structure.

Although, in mammalian cells, long ds iRNA agents can induce theinterferon response which is frequently deleterious, short ds iRNAagents do not trigger the interferon response, at least not to an extentthat is deleterious to the cell and host. The iRNA agents of the presentinvention include molecules which are sufficiently short that they donot trigger the interferon response in mammalian cells. Thus, theadministration of a composition of an iRNA agent (e.g., formulated asdescribed herein) to a mammalian cell can be used to silence expressionof the ApoB gene while circumventing the interferon response. Moleculesthat are short enough that they do not trigger an interferon responseare termed siRNA agents or siRNAs herein. “siRNA agent” or “siRNA” asused herein, refers to an iRNA agent, e.g., a ds iRNA agent or singlestrand RNA agent, that is sufficiently short that it does not induce adeleterious interferon response in a human cell, e.g., it has a duplexedregion of less than 60 but preferably less than 50, 40, or 30 nucleotidepairs.

Moreover, in one embodiment, a mammalian cell is treated with an iRNAagent that disrupts a component of the interferon response, e.g.,dsRNA-activated protein kinase PKR.

The isolated iRNA agents described herein, including ds iRNA agents andsiRNA agents, can mediate silencing of an ApoB gene, e.g., by RNAdegradation. For convenience, such RNA is also referred to herein as theRNA to be silenced. Such a gene is also referred to as a target gene.Preferably, the RNA to be silenced is a gene product of an endogenousApoB gene.

As used herein, the phrase “mediates RNAi” refers to the ability of anagent to silence, in a sequence specific manner, a target gene.“Silencing a target gene” means the process whereby a cell containingand/or secreting a certain product of the target gene when not incontact with the agent, will contain and/or secret at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product whencontacted with the agent, as compared to a similar cell which has notbeen contacted with the agent. Such product of the target gene can, forexample, be a messenger RNA (mRNA), a protein, or a regulatory element.While not wishing to be bound by theory, it is believed that silencingby the agents described herein uses the RNAi machinery or process and aguide RNA, e.g., an siRNA agent of 15 to 30 nucleotide pairs.

As used herein, “the term “complementary” is used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound of the invention and a target RNAmolecule, e.g. an ApoB mRNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 4 nucleotides.

As used herein, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA (e.g., a target ApoB mRNA) if the iRNAagent reduces the production of a protein encoded by the target RNA in acell. The iRNA agent may also be “exactly complementary” (excluding theSRMS containing subunit(s)) to the target RNA, e.g., the target RNA andthe iRNA agent anneal, preferably to form a hybrid made exclusively ofWatson-Crick basepairs in the region of exact complementarity. A“sufficiently complementary” iRNA agent can include an internal region(e.g., of at least 10 nucleotides) that is exactly complementary to atarget ApoB RNA. Moreover, in some embodiments, the iRNA agentspecifically discriminates a single-nucleotide difference. In this case,the iRNA agent only mediates RNAi if exact complementary is found in theregion (e.g., within 7 nucleotides of) the single-nucleotide difference.Preferred iRNA agents will be based on or consist or comprise the senseand antisense sequences provided in Table 1.

As used herein, “essentially identical” when used referring to a firstnucleotide sequence in comparison to a second nucleotide sequence meansthat the first nucleotide sequence is identical to the second nucleotidesequence except for up to one, two or three nucleotide substitutions(e.g. adenosine replaced by uracil). “Essentially retaining the abilityto inhibit ApoB expression in cultured human HepG2 cells”, as usedherein referring to an iRNA agent not identical to but derived from oneof the iRNA agents of Table 1 by deletion, addition or substitution ofnucleotides, means that the derived iRNA agent possesses an inhibitoryactivity lower by not more than 20% inhibition compared to the iRNAagent of Table 1 it was derived from. E.g. an iRNA agent derived from aniRNA agent of Table 1 which lowers the amount of ApoB mRNA present incultured human HepG2 cells by 70% may itself lower the amount of ApoBmRNA present in cultured human HepG2 cells by at least 50% in order tobe considered as essentially retaining the ability to inhibit ApoBexpression in cultured human HepG2 cells. Optionally, an iRNA agent ofthe invention may lower the amount of ApoB mRNA present in culturedhuman HepG2 cells, or the amount of ApoB protein secreted into cellculture supernatant, by at least 50%.

In a typical embodiment, the subject is a mammal such as a cow, horse,mouse, rat, dog, pig, goat, or a primate. In a much preferredembodiment, the subject is a human, e.g., a normal individual or anindividual that has, is diagnosed with, or is predicted to have adisease or disorder.

Because iRNA agent mediated silencing can persist for several days afteradministering the iRNA agent composition, in many instances, it ispossible to administer the composition with a frequency of less thanonce per day, or, for some instances, only once for the entiretherapeutic regimen.

Disorders Associated with ApoB Misexpression

An iRNA agent that targets ApoB, e.g., an iRNA agent described herein,can be used to treat a subject, e.g., a human having or at risk fordeveloping a disease or disorder associated with aberrant or unwantedApoB gene expression, e.g., ApoB overexpression.

For example, an iRNA agent that targets ApoB mRNA can be used to treat alipid-related disorder, such as hypercholesterolemia, e.g., primaryhypercholesterolemia with peripheral vascular disease. Otherlipid-related disorders include coronary artery disease (CAD),myocardial infarction; HDL/LDL cholesterol imbalance; dyslipidemias(e.g., familial combined hyperlipidemia (FCHL) and acquiredhyperlipidemia); hypercholestorolemia; statin-resistanthypercholesterolemia; coronary heart disease (CHD); thrombosis; andatherosclerosis. In one embodiment, the iRNA that targets ApoB mRNA isadministered to a subject suffering from statin-resistant disorder, e.g.statin-resistant hypercholesterolemia. The subject can be one who iscurrently being treated with a statin, one who has been treated with astatin in the past, or one who is unsuited for treatment with a statin.

An iRNA agent targeting ApoB mRNA can be used to treat a human carryinga genetic mutation or polymorphism in the ApoB gene or in theLDL-receptor. For example, the iRNA agent can be used to treat a humandiagnosed as having familial ligand-defective apolipoprotein B-100(FDB), a dominantly inherited disorder of lipoprotein metabolism leadingto hypercholesterolemia and increased proneness to CAD. Plasmacholesterol levels are dramatically elevated in these subjects due toimpaired clearance of LDL particles by defective ApoB/E receptors.

Design and Selection of iRNA Agents

Example 2 hereinbelow shows a gene walk based on sequence prediction wasused to evaluate 81 potential iRNA agents targeting human and mouse ApoBmRNA. Based on the results provided, Table 1 provides active iRNA agentstargeting ApoB. One can readily design and generate other iRNA agentsthat are based on, comprise or consist of one of the active sequencesprovided herein such that at least a portion of an active sequence isincluded in the iRNA agents.

The iRNA agents shown in Example 2 hereinbelow are composed of a sensestrand of 21 nucleotides in length, and an antisense strand of 23nucleotides in length. However, while these lengths may potentially beoptimal, the iRNA agents are not meant to be limited to these lengths.The skilled person is well aware that shorter or longer iRNA agents maybe similarly effective, since, within certain length ranges, theefficacy is rather a function of the nucleotide sequence than strandlength. For example, Yang, D., et al., PNAS 2002, 99:9942-9947,demonstrated similar efficacies for iRNA agents of lengths between 21and 30 base pairs. Others have shown effective silencing of genes byiRNA agents down to a length of approx. 15 base pairs (Byrom, W. M., etal., Inducing RNAi with siRNA Cocktails Generated by RNase III; TechNotes 10(1), Ambion, Inc., Austin, Tex., USA).

Therefore, it is possible and contemplated by the instant invention toselect from the sequences provided in Table 1 a partial sequence ofbetween 15 to 22 nucleotides for the generation of an iRNA agent derivedfrom one of the sequences provided in Table 1. Alternatively, one mayadd one or several nucleotides to one of the sequences provided in Table1, preferably, but not necessarily, in such a fashion that the addednucleotides are complementary to the respective sequence of the targetgene, e.g. ApoB. All such derived iRNA agents are included in the iRNAagents of the present invention, provided they essentially retain theability to inhibit ApoB expression in cultured human HepG2 cells.

Generally, the iRNA agents of the instant invention include a region ofsufficient complementarity to the ApoB gene, and are of sufficientlength in terms of nucleotides, that the iRNA agent, or a fragmentthereof, can mediate down regulation of the ApoB gene. The antisensestrands of the iRNA agents of Table 1 are fully complementary to themRNA sequences of mouse (GenBank Accession number: XM_(—)137955) andhuman (GenBank Accession number: NM_(—)000384) ApoB, and their sensestrands are fully complementary to the antisense strands except for thetwo 3′-terminal nucleotides on the antisense strand. However, it is notnecessary that there be perfect complementarity between the iRNA agentand the target, but the correspondence must be sufficient to enable theiRNA agent, or a cleavage product thereof, to direct sequence specificsilencing, e.g., by RNAi cleavage of an ApoB mRNA.

Therefore, the iRNA agents of the instant invention include agentscomprising a sense strand and antisense strand each comprising asequence of at least 16, 17 or 18 nucleotides which is essentiallyidentical, as defined below, to one of the sequences of Table 1, exceptthat not more than 1, 2 or 3 nucleotides per strand, respectively, havebeen substituted by other nucleotides (e.g. adenosine replaced byuracil), while essentially retaining the ability to inhibit ApoBexpression in cultured human HepG2 cells, as defined below. These agentswill therefore possess at least 15 nucleotides identical to one of thesequences of Table 1, but 1, 2 or 3 base mismatches with respect toeither the target ApoB mRNA sequence or between the sense and antisensestrand are introduced. Mismatches to the target ApoB mRNA sequence,particularly in the antisense strand, are most tolerated in the terminalregions and if present are preferably in a terminal region or regions,e.g., within 6, 5, 4, or 3 nucleotides of a 5′ and/or 3′ terminus, mostpreferably within 6, 5, 4, or 3 nucleotides of the 5′-terminus of thesense strand or the 3′-terminus of the antisense strand. The sensestrand need only be sufficiently complementary with the antisense strandto maintain the overall double stranded character of the molecule.

The antisense strand of an iRNA agent should be equal to or at least,14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. Itshould be equal to or less than 60, 50, 40, or 30, nucleotides inlength. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21nucleotides in length.

The sense strand of an iRNA agent should be equal to or at least 14, 15,16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should beequal to or less than 60, 50, 40, or 30 nucleotides in length. Preferredranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength.

The double stranded portion of an iRNA agent should be equal to or atleast, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50nucleotide pairs in length. It should be equal to or less than 60, 50,40, or 30 nucleotides pairs in length. Preferred ranges are 15-30, 17 to25, 19 to 23, and 19 to 21 nucleotides pairs in length.

It is preferred that the sense and antisense strands be chosen such thatthe iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, an iRNA agent contains sense andantisense strands, preferably paired to contain an overhang, e.g., oneor two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3nucleotides. Most embodiments will have a 3′ overhang. Preferred siRNAagents will have single-stranded overhangs, preferably 3′ overhangs, of1 to 4, or preferably 2 or 3 nucleotides, in length at each end. Theoverhangs can be the result of one strand being longer than the other,or the result of two strands of the same length being staggered. 5′-endsare preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe siRNA agent range discussed above. siRNA agents can resemble inlength and structure the natural Dicer processed products from longdsRNAs. Embodiments in which the two strands of the siRNA agent arelinked, e.g., covalently linked are also included. Hairpin, or othersingle strand structures which provide the required double strandedregion, and preferably a 3′ overhang are also within the invention.

Much of the discussion below refers to single strand molecules. In manyembodiments of the invention a ds iRNA agent, e.g., a partially ds iRNAagent, is required or preferred. Thus, it is understood that that doublestranded structures (e.g. where two separate molecules are contacted toform the double stranded region or where the double stranded region isformed by intramolecular pairing (e.g., a hairpin structure)) made ofthe single stranded structures described below are within the invention.Preferred lengths are described elsewhere herein.

Evaluation of Candidate iRNA Agents

A candidate iRNA agent can be evaluated for its ability to downregulatetarget gene expression. For example, a candidate iRNA agent can beprovided, and contacted with a cell, e.g. a HepG2 cell, that expressesthe target gene, e.g., the ApoB gene, either endogenously or because ithas been transfected with a construct from which ApoB can be expressed.The level of target gene expression prior to and following contact withthe candidate iRNA agent can be compared, e.g. on an mRNA or proteinlevel. If it is determined that the amount of RNA or protein expressedfrom the target gene is lower following contact with the iRNA agent,then it can be concluded that the iRNA agent downregulates target geneexpression. The level of target ApoB RNA or ApoB protein in the cell canbe determined by any method desired. For example, the level of targetRNA can be determined by Northern blot analysis, reverse transcriptioncoupled with polymerase chain reaction (RT-PCR), or RNAse protectionassay. The level of protein can be determined, for example, by Westernblot analysis.

Stability Testing, Modification, and Retesting of iRNA Agents

A candidate iRNA agent can be evaluated with respect to stability, e.g,its susceptibility to cleavage by an endonuclease or exonuclease, suchas when the iRNA agent is introduced into the body of a subject. Methodscan be employed to identify sites that are susceptible to modification,particularly cleavage, e.g., cleavage by a component found in the bodyof a subject.

When sites susceptible to cleavage are identified, a further iRNA agentcan be designed and/or synthesized wherein the potential cleavage siteis made resistant to cleavage, e.g. by introduction of a 2′-modificationon the site of cleavage, e.g. a 2′-O-methyl group. This further iRNAagent can be retested for stability, and this process may be iterateduntil an iRNA agent is found exhibiting the desired stability.

In Vivo Testing

An iRNA agent identified as being capable of inhibiting ApoB geneexpression can be tested for functionality in vivo in an animal model(e.g., in a mammal, such as in mouse or rat). For example, the iRNAagent can be administered to an animal, and the iRNA agent evaluatedwith respect to its biodistribution, stability, and its ability toinhibit ApoB gene expression.

The iRNA agent can be administered directly to the target tissue, suchas by injection, or the iRNA agent can be administered to the animalmodel in the same manner that it would be administered to a human

The iRNA agent can also be evaluated for its intracellular distribution.The evaluation can include determining whether the iRNA agent was takenup into the cell. The evaluation can also include determining thestability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNAagent in vivo can be facilitated by use of an iRNA agent conjugated to atraceable marker (e.g., a fluorescent marker such as fluorescein; aradioactive label, such as ³⁵S, ³²P, ³³P, or ³H; gold particles; orantigen particles for immunohistochemistry).

An iRNA agent useful for monitoring biodistribution can lack genesilencing activity in vivo. For example, the iRNA agent can target agene not present in the animal (e.g., an iRNA agent injected into mousecan target luciferase), or an iRNA agent can have a non-sense sequence,which does not target any gene, e.g., any endogenous gene).Localization/biodistribution of the iRNA can be monitored, e.g. by atraceable label attached to the iRNA agent, such as a traceable agentdescribed above

The iRNA agent can be evaluated with respect to its ability to downregulate ApoB gene expression. Levels of ApoB gene expression in vivocan be measured, for example, by in situ hybridization, or by theisolation of RNA from tissue prior to and following exposure to the iRNAagent. Where the animal needs to be sacrificed in order to harvest thetissue, an untreated control animal will serve for comparison. TargetApoB mRNA can be detected by any desired method, including but notlimited to RT-PCR, Northern blot, branched-DNA assay, or RNAaseprotection assay. Alternatively, or additionally, ApoB gene expressioncan be monitored by performing Western blot analysis on tissue extractstreated with the iRNA agent.

iRNA Chemistry

Described herein are isolated iRNA agents, e.g., RNA molecules,(double-stranded; single-stranded) that mediate RNAi to inhibitexpression of ApoB.

RNA agents discussed herein include otherwise unmodified RNA as well asRNA which have been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The art hasreferred to rare or unusual, but naturally occurring, RNAs as modifiedRNAs, see, e.g., Limbach et al., (1994) Nucleic Acids Res. 22:2183-2196. Such rare or unusual RNAs, often termed modified RNAs(apparently because the are typically the result of apost-transcriptional modification) are within the term unmodified RNA,as used herein. Modified RNA as used herein refers to a molecule inwhich one or more of the components of the nucleic acid, namely sugars,bases, and phosphate moieties, are different from that which occur innature, preferably different from that which occurs in the human body.While they are referred to as modified “RNAs,” they will of course,because of the modification, include molecules which are not RNAs.Nucleoside surrogates are molecules in which the ribophosphate backboneis replaced with a non-ribophosphate construct that allows the bases tothe presented in the correct spatial relationship such thathybridization is substantially similar to what is seen with aribophosphate backbone, e.g., non-charged mimics of the ribophosphatebackbone. Examples of all of the above are discussed herein.

Modifications described herein can be incorporated into anydouble-stranded RNA and RNA-like molecule described herein, e.g., aniRNA agent. It may be desirable to modify one or both of the antisenseand sense strands of an iRNA agent. As nucleic acids are polymers ofsubunits or monomers, many of the modifications described below occur ata position which is repeated within a nucleic acid, e.g., a modificationof a base, or a phosphate moiety, or the non-linking O of a phosphatemoiety. In some cases the modification will occur at all of the subjectpositions in the nucleic acid but in many, and in fact in most, cases itwill not. By way of example, a modification may only occur at a 3′ or 5′terminal position, may only occur in a terminal region, e.g. at aposition on a terminal nucleotide or in the last 2, 3, 4, 5, or 10nucleotides of a strand. A modification may occur in a double strandregion, a single strand region, or in both. E.g., a phosphorothioatemodification at a non-linking O position may only occur at one or bothtermini, may only occur in a terminal regions, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand, or may occur in double strand and single strand regions,particularly at termini. Similarly, a modification may occur on thesense strand, antisense strand, or both. In some cases, the sense andantisense strand will have the same modifications or the same class ofmodifications, but in other cases the sense and antisense strand willhave different modifications, e.g., in some cases it may be desirable tomodify only one strand, e.g. the sense strand.

Two prime objectives for the introduction of modifications into iRNAagents is their stabilization towards degradation in biologicalenvironments and the improvement of pharmacological properties, e.g.pharmacodynamic properties, which are further discussed below. Othersuitable modifications to a sugar, base, or backbone of an iRNA agentare described in co-owned PCT Application No. PCT/US2004/01193, filedJan. 16, 2004. An iRNA agent can include a non-naturally occurring base,such as the bases described in co-owned PCT Application No.PCT/US2004/011822, filed Apr. 16, 2004. An iRNA agent can include anon-naturally occurring sugar, such as a non-carbohydrate cyclic carriermolecule. Exemplary features of non-naturally occurring sugars for usein iRNA agents are described in co-owned PCT Application No.PCT/US2004/11829 filed Apr. 16, 2003.

An iRNA agent can include an internucleotide linkage (e.g., the chiralphosphorothioate linkage) useful for increasing nuclease resistance. Inaddition, or in the alternative, an iRNA agent can include a ribosemimic for increased nuclease resistance. Exemplary internucleotidelinkages and ribose mimics for increased nuclease resistance aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can include ligand-conjugated monomer subunits andmonomers for oligonucleotide synthesis. Exemplary monomers are describedin co-owned U.S. application Ser. No. 10/916,185, filed on Aug. 10,2004.

An iRNA agent can have a ZXY structure, such as is described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

An iRNA agent can be complexed with an amphipathic moiety. Exemplaryamphipathic moieties for use with iRNA agents are described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

The sense and antisense sequences of an iRNA agent can be palindromic.Exemplary features of palindromic iRNA agents are described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

In another embodiment, the iRNA agent can be complexed to a deliveryagent that features a modular complex. The complex can include a carrieragent linked to one or more of (preferably two or more, more preferablyall three of): (a) a condensing agent (e.g., an agent capable ofattracting, e.g., binding, a nucleic acid, e.g., through ionic orelectrostatic interactions); (b) a fusogenic agent (e.g., an agentcapable of fusing and/or being transported through a cell membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type. iRNA agents complexed to a delivery agent aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can have non-canonical pairings, such as between the senseand antisense sequences of the iRNA duplex. Exemplary features ofnon-canonical iRNA agents are described in co-owned PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004.

Enhanced Nuclease Resistance

An iRNA agent, e.g., an iRNA agent that targets ApoB, can have enhancedresistance to nucleases. One way to increase resistance is to identifycleavage sites and modify such sites to inhibit cleavage. For example,the dinucleotides 5′-UA-3′, 5′-UG-3′, 5′-CA-3′, 5′-UU-3′, or 5′-CC-3′can serve as cleavage sites, as described in co-owned and co-pendingapplications U.S. 60/574,744 and PCT/US2005/018931.

For increased nuclease resistance and/or binding affinity to the target,an iRNA agent, e.g., the sense and/or antisense strands of the iRNAagent, can include, for example, 2′-modified ribose units and/orphosphorothioate linkages. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality. Preferredsubstitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and2′-fluoro.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

In certain embodiments, all the pyrimidines of an iRNA agent carry a2′-modification, and the iRNA agent therefore has enhanced resistance toendonucleases. Enhanced nuclease resistance can also be achieved bymodifying the 5′ nucleotide, resulting, for example, in at least one5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein the uridine is a2′-modified nucleotide; at least one 5′-uridine-guanine-3′ (5′-UG-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; atleast one 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide; at least one5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide; or at least one 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide. The iRNA agent can include at least 2, at least 3, at least4 or at least 5 of such dinucleotides. Preferably, the 5′-mostpyrimidines in all occurrences of the sequence motifs 5′-UA-3′,5′-CA-3′, 5′-UU-3′, and 5′-UG-3′ are 2′-modified nucleotides. Morepreferably, all pyrimidines in the sense strand are 2′-modifiednucleotides, and the 5′-most pyrimidines in all occurrences of thesequence motifs 5′-UA-3′ and 5′-CA-3′. Most preferably, all pyrimidinesin the sense strand are 2′-modified nucleotides, and the 5′-mostpyrimidines in all occurrences of the sequence motifs 5′-UA-3′,5′-CA-3′, 5′-UU-3′, and 5′-UG-3′ are 2′-modified nucleotides in theantisense strand. The latter patterns of modifications have been shownby the instant inventors to maximize the contribution of the nucleotidemodifications to the stabilization of the overall molecule towardsnuclease degradation, while minimizing the overall number ofmodifications required to a desired stability, see co-owned andco-pending PCT/US2005/018931, hereby incorporated herein by reference inits entirety.

The inclusion of furanose sugars in the oligonucleotide backbone canalso decrease endonucleolytic cleavage. An iRNA agent can be furthermodified by including a 3′ cationic group, or by inverting thenucleoside at the 3′-terminus with a 3′-3′ linkage. In anotheralternative, the 3′-terminus can be blocked with an aminoalkyl group,e.g., a 3′ C5-aminoalkyl dT. Other 3′ conjugates can inhibit 3′-5′exonucleolytic cleavage. While not being bound by theory, a 3′conjugate, such as naproxen or ibuprofen, may inhibit exonucleolyticcleavage by sterically blocking the exonuclease from binding to the3′-end of oligonucleotide. Even small alkyl chains, aryl groups, orheterocyclic conjugates or modified sugars (D-ribose, deoxyribose,glucose etc.) can block 3′-5′-exonucleases.

Similarly, 5′ conjugates can inhibit 5′-3′ exonucleolytic cleavage.While not being bound by theory, a 5′ conjugate, such as naproxen oribuprofen, may inhibit exonucleolytic cleavage by sterically blockingthe exonuclease from binding to the 5′-end of oligonucleotide. Evensmall alkyl chains, aryl groups, or heterocyclic conjugates or modifiedsugars (D-ribose, deoxyribose, glucose etc.) can block3′-5′-exonucleases.

An iRNA agent can have increased resistance to nucleases when a duplexediRNA agent includes a single-stranded nucleotide overhang on at leastone end. In preferred embodiments, the nucleotide overhang includes 1 to4, preferably 2 to 3, unpaired nucleotides. In a preferred embodiment,the unpaired nucleotide of the single-stranded overhang that is directlyadjacent to the terminal nucleotide pair contains a purine base, and theterminal nucleotide pair is a G-C pair, or at least two of the last fourcomplementary nucleotide pairs are G-C pairs. In further embodiments,the nucleotide overhang may have 1 or 2 unpaired nucleotides, and in anexemplary embodiment the nucleotide overhang is 5′-GC-3′. In preferredembodiments, the nucleotide overhang is on the 3′-end of the antisensestrand. In one embodiment, the iRNA agent includes the motif 5′-CGC-3′on the 3′-end of the antisense strand, such that a 2-nt overhang5′-GC-3′ is formed.

Thus, an iRNA agent can include monomers which have been modified so asto inhibit degradation, e.g., by nucleases, e.g., endonucleases orexonucleases, found in the body of a subject. These monomers arereferred to herein as NRMs, or Nuclease Resistance promoting Monomers ormodifications. In many cases these modifications will modulate otherproperties of the iRNA agent as well, e.g., the ability to interact witha protein, e.g., a transport protein, e.g., serum albumin, or a memberof the RISC, or the ability of the first and second sequences to form aduplex with one another or to form a duplex with another sequence, e.g.,a target molecule.

While not wishing to be bound by theory, it is believed thatmodifications of the sugar, base, and/or phosphate backbone in an iRNAagent can enhance endonuclease and exonuclease resistance, and canenhance interactions with transporter proteins and one or more of thefunctional components of the RISC complex. Preferred modifications arethose that increase exonuclease and endonuclease resistance and thusprolong the half-life of the iRNA agent prior to interaction with theRISC complex, but at the same time do not render the iRNA agent inactivewith respect to its intended activity as a target mRNA cleavagedirecting agent. Again, while not wishing to be bound by any theory, itis believed that placement of the modifications at or near the 3′ and/or5′-end of antisense strands can result in iRNA agents that meet thepreferred nuclease resistance criteria delineated above. Again, stillwhile not wishing to be bound by any theory, it is believed thatplacement of the modifications at e.g., the middle of a sense strand canresult in iRNA agents that are relatively less likely to show off-targeteffects.

Modifications that can be useful for producing iRNA agents that meet thepreferred nuclease resistance criteria delineated above can include oneor more of the following chemical and/or stereochemical modifications ofthe sugar, base, and/or phosphate backbone:

(i) chiral (S_(P)) thioates. Thus, preferred NRMs include nucleotidedimers with an enriched or pure for a particular chiral form of amodified phosphate group containing a heteroatom at the nonbridgingposition, e.g., Sp or Rp, at the position X, where this is the positionnormally occupied by the oxygen. The atom at X can also be S, Se, Nr₂,or Br₃. When X is S, enriched or chirally pure Sp linkage is preferred.Enriched means at least 70, 80, 90, 95, or 99% of the preferred form.Such NRMs are discussed in more detail below;

(ii) attachment of one or more cationic groups to the sugar, base,and/or the phosphorus atom of a phosphate or modified phosphate backbonemoiety. Thus, preferred NRMs include monomers at the terminal positionderivatized at a cationic group. As the 5′-end of an antisense sequenceshould have a terminal —OH or phosphate group this NRM is preferably notused at the 5′-end of an antisense sequence. The group should beattached at a position on the base which minimizes interference with Hbond formation and hybridization, e.g., away form the face whichinteracts with the complementary base on the other strand, e.g, at the5′ position of a pyrimidine or a 7-position of a purine. These arediscussed in more detail below;

(iii) nonphosphate linkages at the termini. Thus, preferred NRMs includeNon-phosphate linkages, e.g., a linkage of 4 atoms which confers greaterresistance to cleavage than does a phosphate bond. Examples include 3′CH2-NCH₃—O—CH₂-5′ and 3′ CH₂—NH—(O═)—CH₂-5′;

(iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Thus,preferred NRM's can included these structures;

(v) L-RNA, 2′-5′ linkages, inverted linkages, a-nucleosides. Thus, otherpreferred NRM's include: L nucleosides and dimeric nucleotides derivedfrom L-nucleosides; 2′-5′ phosphate, non-phosphate and modifiedphosphate linkages (e.g., thiophosphates, phosphoramidates andboronophosphates); dimers having inverted linkages, e.g., 3′-3′ or 5′-5′linkages; monomers having an alpha linkage at the 1′ site on the sugar,e.g., the structures described herein having an alpha linkage;

(vi) conjugate groups. Thus, preferred NRM's can include, e.g., atargeting moiety or a conjugated ligand described herein conjugated withthe monomer, e.g., through the sugar, base, or backbone;

(vi) abasic linkages. Thus, preferred NRM's can include an abasicmonomer, e.g., an abasic monomer as described herein (e.g., anucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclicaromatic monomer as described herein; and

(vii) 5′-phosphonates and 5′-phosphate prodrugs. Thus, preferred NRM'sinclude monomers, preferably at the terminal position, e.g., the 5′position, in which one or more atoms of the phosphate group isderivatized with a protecting group, which protecting group or groups,are removed as a result of the action of a component in the subject'sbody, e.g, a carboxyesterase or an enzyme present in the subject's body.E.g., a phosphate prodrug in which a carboxy esterase cleaves theprotected molecule resulting in the production of a thioate anion whichattacks a carbon adjacent to the O of a phosphate and resulting in theproduction of an unprotected phosphate.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent. As some NRMsinterfere with hybridization the total number incorporated, should besuch that acceptable levels of iRNA agent duplex formation aremaintained.

In some embodiments NRM modifications are introduced into the terminalcleavage site or in the cleavage region of a sequence (a sense strand orsequence) which does not target a desired sequence or gene in thesubject. This can reduce off-target silencing.

Nuclease resistant modifications include some which can be placed onlyat the terminus and others which can go at any position. Generally themodifications that can inhibit hybridization so it is preferably to usethem only in terminal regions, and preferable to not use them at thecleavage site or in the cleavage region of an sequence which targets asubject sequence or gene. The can be used anywhere in a sense sequence,provided that sufficient hybridization between the two sequences of theiRNA agent is maintained. In some embodiments it is desirable to put theNRM at the cleavage site or in the cleavage region of a sequence whichdoes not target a subject sequence or gene, as it can minimizeoff-target silencing.

In addition, an iRNA agent described herein can have an overhang whichdoes not form a duplex structure with the other sequence of the iRNAagent—it is an overhang, but it does hybridize, either with itself, orwith another nucleic acid, other than the other sequence of the iRNAagent.

In most cases, the nuclease-resistance promoting modifications will bedistributed differently depending on whether the sequence will target asequence in the subject (often referred to as an antisense sequence) orwill not target a sequence in the subject (often referred to as a sensesequence). If a sequence is to target a sequence in the subject,modifications which interfere with or inhibit endonuclease cleavageshould not be inserted in the region which is subject to RISC mediatedcleavage, e.g., the cleavage site or the cleavage region (As describedin Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated byreference). Cleavage of the target occurs about in the middle of a 20 or21 nt guide RNA, or about 10 or 11 nucleotides upstream of the firstnucleotide which is complementary to the guide sequence. As used hereincleavage site refers to the nucleotide on either side of the cleavagesite, on the target or on the iRNA agent strand which hybridizes to it.Cleavage region means an nucleotide with 1, 2, or 3 nucleotides of thecleave site, in either direction.)

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sequence which targets or a sequence which does not target asequence in the subject.

Tethered Ligands

The properties of an iRNA agent, including its pharmacologicalproperties, can be influenced and tailored, for example, by theintroduction of ligands, e.g. tethered ligands.

A wide variety of entities, e.g., ligands, can be tethered to an iRNAagent, e.g., to the carrier of a ligand-conjugated monomer subunit.Examples are described below in the context of a ligand-conjugatedmonomer subunit but that is only preferred, entities can be coupled atother points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe carrier. In preferred embodiments, the ligand is attached to thecarrier via an intervening tether. The ligand or tethered ligand may bepresent on the ligand-conjugated monomer\when the ligand-conjugatedmonomer is incorporated into the growing strand. In some embodiments,the ligand may be incorporated into a “precursor” ligand-conjugatedmonomer subunit after a “precursor” ligand-conjugated monomer subunithas been incorporated into the growing strand. For example, a monomerhaving, e.g., an amino-terminated tether, e.g., TAP—(CH₂)_(n)NH₂ may beincorporated into a growing sense or antisense strand. In a subsequentoperation, i.e., after incorporation of the precursor monomer subunitinto the strand, a ligand having an electrophilic group, e.g., apentafluorophenyl ester or aldehyde group, can subsequently be attachedto the precursor ligand-conjugated monomer by coupling the electrophilicgroup of the ligand with the terminal nucleophilic group of theprecursor ligand-conjugated monomer subunit tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g, molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases. Generalexamples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin,diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin,Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g.,folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins,protein binding agents, integrin targeting molecules, polycationics,peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid); amino acid, or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polyaminoacid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationicmoieties, e.g., cationic lipid, cationic porphyrin, quaternary salt of apolyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a liver cell ora cell of the jejunum. A targeting group can be a thyrotropin,melanotropin, lectin, glycoprotein, surfactant protein A, Mucincarbohydrate, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent fucose, glycosylated polyaminoacids, multivalent galactose,transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid,cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or anRGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., estersand ethers thereof, e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈,C₁₉, or C₂₀ alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol,1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group,hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group,palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP,or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., liver tissue, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and petomimetics can target cancercells, in particular cells that exhibit an I_(v)θ₃ integrin. Thus, onecould use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the I_(v)−θ₃ integrinligand. Generally, such ligands can be used to control proliferatingcells and angiogeneis. Preferred conjugates of this type include an iRNAagent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancergene described herein.

A targeting agent that incorporates a sugar, e.g., galactose and/oranalogues thereof, is particularly useful. These agents target, inparticular, the parenchymal cells of the liver. For example, a targetingmoiety can include more than one or preferably two or three galactosemoieties, spaced about 15 angstroms from each other. The targetingmoiety can alternatively be lactose (e.g., three lactose moieties),which is glucose coupled to a galactose. The targeting moiety can alsobe N-Acetyl-Galactosamine, N—Ac-Glucosamine. A mannose ormannose-6-phosphate targeting moiety can be used for macrophagetargeting.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

iRNA Conjugates

An iRNA agent can be coupled, e.g., covalently coupled, to a secondagent. For example, an iRNA agent used to treat a particular disorder,such as a lipid disorder, can be coupled to a second therapeutic agent,e.g., an agent other than the iRNA agent. The second therapeutic agentcan be one which is directed to the treatment of the same disorder.

5′-Phosphate Modifications

In preferred embodiments, iRNA agents are 5′ phosphorylated or include aphosphoryl analog at the 5′ prime terminus. 5′-phosphate modificationsof the antisense strand include those which are compatible with RISCmediated gene silencing. Suitable modifications include:5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

The sense strand can be modified in order to inactivate the sense strandand prevent formation of an active RISC, thereby potentially reducingoff-target effects. This can be accomplished by a modification whichprevents 5′-phosphorylation of the sense strand, e.g., by modificationwith a 5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATPrequirements and small interfering RNA structure in the RNA interferencepathway. Cell 107, 309-321.) Other modifications which preventphosphorylation can also be used, e.g., simply substituting the 5′-OH byH rather than O-Me. Alternatively, a large bulky group may be added tothe 5′-phosphate turning it into a phosphodiester linkage.

Delivery of iRNA Agents to Tissues and Cells

Targeting to the Liver

The iRNA agent that targets ApoB can be targeted to the liver, forexample by associating, e.g., conjugating the iRNA agent to a lipophilicmoiety, e.g., a lipid, oleyl, retinyl, or cholesteryl residue.Conjugation to cholesterol is preferred. Other lipophilic moieties thatcan be associated, e.g., conjugated with the iRNA agent include cholicacid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.Alternatively, the iRNA agent can be targeted to the liver byassociating, e.g., conjugating, the iRNA agent to a low-densitylipoprotein (LDL), e.g., a lactosylated LDL, or the iRNA agent can betargeted to the liver by associating, e.g., conjugating, the iRNA agentto a polymeric carrier complex with sugar residues.

The iRNA agent can be targeted to the liver by associating, e.g.,conjugating, the iRNA agent to a liposome complexed with sugar residues.A targeting agent that incorporates a sugar, e.g., galactose and/oranalogues thereof, is particularly useful. These agents target, inparticular, the parenchymal cells of the liver. Preferably, thetargeting moiety includes more than one galactose moiety, morepreferably two or three. Most preferably, the targeting moiety includesthree galactose moieties, e.g., spaced about 15 angstroms from eachother. The targeting moiety can be lactose. A lactose is a glucosecoupled to a galactose. Preferably, the targeting moiety includes threelactoses. The targeting moiety can also be N-Acetyl-Galactosamine,N—Ac-Glucosamine. A mannose, or mannose-6-phosphate targeting moiety canbe used for macrophage targeting.

An iRNA agent can also be targeted to the liver by association with alow-density lipoprotein (LDL), such as lactosylated LDL. Polymericcarriers complexed with sugar residues can also function to target iRNAagents to the liver.

The targeting agent can be linked directly, e.g., covalently or noncovalently, to the iRNA agent, or to another delivery or formulationmodality, e.g., a liposome. E.g., the iRNA agents with or without atargeting moiety can be incorporated into a delivery modality, e.g., aliposome, with or without a targeting moiety.

The iRNA agent that targets ApoB can be targeted to the liver, forexample by associating, e.g., conjugating the iRNA agent, to a serumalbumin (SA) molecule, e.g., a human serum albumin (HSA) molecule, or afragment thereof. The iRNA agent or composition thereof can have anaffinity for an SA, e.g., HSA, which is sufficiently high such that itslevels in the liver are at least 10, 20, 30, 50, or 100% greater in thepresence of SA, e.g., HSA, or is such that addition of exogenous SA willincrease delivery to the liver. These criteria can be measured, e.g., bytesting distribution in a mouse in the presence or absence of exogenousmouse or human SA.

The SA, e.g., HSA, targeting agent can be linked directly, e.g.,covalently or non-covalently, to the iRNA agent, or to another deliveryor formulation modality, e.g., a liposome. E.g., the iRNA agents with orwithout a targeting moiety can be incorporated into a delivery modality,e.g., a liposome, with or without a targeting moiety.

Transport of iRNA Agents into Cells

Not wishing to be bound by any theory, the chemical similarity betweencholesterol-conjugated iRNA agents and certain constituents oflipoproteins (e.g. cholesterol, cholesteryl esters, phospholipids) maylead to the association of iRNA agents with lipoproteins (e.g. LDL, HDL)in blood and/or the interaction of the iRNA agent with cellularcomponents having an affinity for cholesterol, e.g. components of thecholesterol transport pathway. Lipoproteins as well as theirconstituents are taken up and processed by cells by various active andpassive transport mechanisms, for example, without limitation,endocytosis of LDL-receptor bound LDL, endocytosis of oxidized orotherwise modified LDLs through interaction with Scavenger receptor A,Scavenger receptor B1-mediated uptake of HDL cholesterol in the liver,pinocytosis, or transport of cholesterol across membranes by ABC(ATP-binding cassette) transporter proteins, e.g. ABC-A1, ABC-G1 orABC-G4. Hence, cholesterol-conjugated iRNA agents could enjoyfacilitated uptake by cells possessing such transport mechanisms, e.g.cells of the liver. As such, the present invention provides evidence andgeneral methods for targeting IRNA agents to cells expressing certaincell surface components, e.g. receptors, by conjugating a natural ligandfor such component (e.g. cholesterol) to the iRNA agent, or byconjugating a chemical moiety (e.g. cholesterol) to the iRNA agent whichassociates with or binds to a natural ligand for the component (e.g.LDL, HDL).

Other Embodiments

An RNA, e.g., an iRNA agent, can be produced in a cell in vivo, e.g.,from exogenous DNA templates that are delivered into the cell. Forexample, the DNA templates can be inserted into vectors and used as genetherapy vectors. Gene therapy vectors can be delivered to a subject by,for example, intravenous injection, local administration (U.S. Pat. No.5,328,470), or by stereotactic injection (see, e.g., Chen et al., Proc.Natl. Acad. Sci. USA 91:3054-3057, 1994). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. The DNA templates, for example, caninclude two transcription units, one that produces a transcript thatincludes the top strand of an iRNA agent and one that produces atranscript that includes the bottom strand of an iRNA agent. When thetemplates are transcribed, the iRNA agent is produced, and processedinto siRNA agent fragments that mediate gene silencing.

Physiological Effects

The iRNA agents described herein can be designed such that determiningtherapeutic toxicity is made easier by the complementarity of the iRNAagent with both a human and a non-human animal sequence. By thesemethods, an iRNA agent can consist of a sequence that is fullycomplementary to a nucleic acid sequence from a human and a nucleic acidsequence from at least one non-human animal, e.g., a non-human mammal,such as a rodent, ruminant or primate. For example, the non-human mammalcan be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus,Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence ofthe iRNA agent could be complementary to sequences within homologousgenes, e.g., oncogenes or tumor suppressor genes, of the non-humanmammal and the human. By determining the toxicity of the iRNA agent inthe non-human mammal, one can extrapolate the toxicity of the iRNA agentin a human. For a more strenuous toxicity test, the iRNA agent can becomplementary to a human and more than one, e.g., two or three or more,non-human animals.

The methods described herein can be used to correlate any physiologicaleffect of an iRNA agent on a human, e.g., any unwanted effect, such as atoxic effect, or any positive, or desired effect.

iRNA Production

An iRNA can be produced, e.g., in bulk, by a variety of methods.Exemplary methods include: organic synthesis and RNA cleavage, e.g., invitro cleavage.

Organic Synthesis.

An iRNA can be made by separately synthesizing each respective strand ofa double-stranded RNA molecule. The component strands can then beannealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB(Uppsala Sweden), can be used to produce a large amount of a particularRNA strand for a given iRNA. The OligoPilotII reactor can efficientlycouple a nucleotide using only a 1.5 molar excess of a phosphoramiditenucleotide. To make an RNA strand, ribonucleotides amidites are used.Standard cycles of monomer addition can be used to synthesize theoligonucleotide strands for the iRNA. Typically, the two complementarystrands are produced separately and then annealed, e.g., after releasefrom the solid support and deprotection.

Organic synthesis can be used to produce a discrete iRNA species. Thecomplementarity of the species to the ApoB gene can be preciselyspecified. For example, the species may be complementary to a regionthat includes a polymorphism, e.g., a single nucleotide polymorphism.Further the location of the polymorphism can be precisely defined. Insome embodiments, the polymorphism is located in an internal region,e.g., at least 4, 5, 7, or 9 nucleotides from one or both of thetermini.

dsRNA Cleavage.

iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can bemediated in vitro or in vivo. For example, to produce iRNAs by cleavagein vitro, the following method can be used:

In vitro transcription. dsRNA is produced by transcribing a nucleic acid(DNA) segment in both directions. For example, the HiScribe™ RNAitranscription kit (New England Biolabs) provides a vector and a methodfor producing a dsRNA for a nucleic acid segment that is cloned into thevector at a position flanked on either side by a T7 promoter. Separatetemplates are generated for T7 transcription of the two complementarystrands for the dsRNA. The templates are transcribed in vitro byaddition of T7 RNA polymerase and dsRNA is produced. Similar methodsusing PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) canalso be used. In one embodiment, RNA generated by this method iscarefully purified to remove endotoxins that may contaminatepreparations of the recombinant enzymes.

In Vitro Cleavage.

dsRNA is cleaved in vitro into iRNAs, for example, using a Dicer orcomparable RNAse III-based activity. For example, the dsRNA can beincubated in an in vitro extract from Drosophila or using purifiedcomponents, e.g. a purified RNAse or RISC complex. See, e.g., Ketting etal. Genes Dev 2001 Oct. 15; 15(20):2654-9. and Hammond Science 2001 Aug.10; 293(5532):1146-50.

dsRNA cleavage generally produces a plurality of iRNA species, eachbeing a particular 21 to 23 nt fragment of a source dsRNA molecule. Forexample, iRNAs that include sequences complementary to overlappingregions and adjacent regions of a source dsRNA molecule may be present.

Regardless of the method of synthesis, the iRNA preparation can beprepared in a solution (e.g., an aqueous and/or organic solution) thatis appropriate for formulation. For example, the iRNA preparation can beprecipitated and redissolved in pure double-distilled water, andlyophilized. The dried iRNA can then be resuspended in a solutionappropriate for the intended formulation process.

Synthesis of modified and nucleotide surrogate iRNA agents is discussedbelow.

Formulation

The iRNA agents described herein can be formulated for administration toa subject.

For ease of exposition, the formulations, compositions, and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions, and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In someexamples, the composition is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA is in an aqueous phase, e.g., in asolution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAcomposition is formulated in a manner that is compatible with theintended method of administration.

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

An iRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent,e.g., a second iRNA agent that can mediate RNAi with respect to a secondgene, or with respect to the same gene. Still other preparations caninclude at least three, five, ten, twenty, fifty, or a hundred or moredifferent iRNA species. Such iRNAs can mediated RNAi with respect to asimilar number of different genes.

In one embodiment, the iRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than an RNA or a DNA).

In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent,or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which canbe processed into an siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) is formulated to target a particular cell. For example, aliposome or particle or other structure that includes a iRNA can alsoinclude a targeting moiety that recognizes a specific molecule on atarget cell. The targeting moiety can be a molecule with a specificaffinity for a target cell. Targeting moieties can include antibodiesdirected against a protein found on the surface of a target cell, or theligand or a receptor-binding portion of a ligand for a molecule found onthe surface of a target cell.

In one embodiment, the targeting moiety is attached to a liposome. Forexample, U.S. Pat. No. 6,245,427 describes a method for targeting aliposome using a protein or peptide. In another example, a cationiclipid component of the liposome is derivatized with a targeting moiety.For example, WO 96/37194 describes convertingN-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimideactivated ester. The product was then coupled to an RGD peptide.Additional targeting methods are described elsewhere herein.

Treatment Methods and Routes of Delivery

A composition that includes an iRNA agent, e.g., an iRNA agent thattargets ApoB, can be delivered to a subject by a variety of routes.Exemplary routes include intrathecal, parenchymal, intravenous, nasal,oral, and ocular delivery. An iRNA agent can be incorporated intopharmaceutical compositions suitable for administration. For example,compositions can include one or more species of an iRNA agent and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, intranasal,transdermal), oral or parenteral. Parenteral administration includesintravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, or intrathecal or intraventricular administration.

The route of delivery can be dependent on the disorder of the patient.

In general, an iRNA agent can be administered by any suitable method. Asused herein, topical delivery can refer to the direct application of aniRNA agent to any surface of the body, including the eye, a mucousmembrane, surfaces of a body cavity, or to any internal surface.Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, sprays, and liquids.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Topicaladministration can also be used as a means to selectively deliver theiRNA agent to the epidermis or dermis of a subject, or to specificstrata thereof, or to an underlying tissue.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

An iRNA agent can be administered to a subject by pulmonary delivery.Pulmonary delivery compositions can be delivered by inhalation by thepatient of a dispersion so that the composition, preferably iRNA, withinthe dispersion can reach the lung where it can be readily absorbedthrough the alveolar region directly into blood circulation. Pulmonarydelivery can be effective both for systemic delivery and for localizeddelivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. An iRNAcomposition may be stably stored as lyophilized or spray-dried powdersby itself or in combination with suitable powder carriers. The deliveryof a composition for inhalation can be mediated by a dosing timingelement which can include a timer, a dose counter, time measuringdevice, or a time indicator which when incorporated into the deviceenables dose tracking, compliance monitoring, and/or dose triggering toa patient during administration of the aerosol medicament.

An iRNA agent can be modified such that it is capable of traversing theblood brain barrier. For example, the iRNA agent can be conjugated to amolecule that enables the agent to traverse the barrier. Such modifiediRNA agents can be administered by any desired method, such as byintraventricular or intramuscular injection, or by pulmonary delivery,for example.

An iRNA agent can be administered ocularly, such as to treat retinaldisorder, e.g., a retinopathy. For example, the pharmaceuticalcompositions can be applied to the surface of the eye or nearby tissue,e.g., the inside of the eyelid. They can be applied topically, e.g., byspraying, in drops, as an eyewash, or an ointment. Ointments ordroppable liquids may be delivered by ocular delivery systems known inthe art such as applicators or eye droppers. Such compositions caninclude mucomimetics such as hyaluronic acid, chondroitin sulfate,hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives suchas sorbic acid, EDTA or benzylchronium chloride, and the usualquantities of diluents and/or carriers. The pharmaceutical compositioncan also be administered to the interior of the eye, and can beintroduced by a needle or other delivery device which can introduce itto a selected area or structure. The composition containing the iRNAagent can also be applied via an ocular patch.

An iRNA agent can be administered by an oral or nasal delivery. Forexample, drugs administered through these membranes have a rapid onsetof action, provide therapeutic plasma levels, avoid first pass effect ofhepatic metabolism, and avoid exposure of the drug to the hostilegastrointestinal (GI) environment. Additional advantages include easyaccess to the membrane sites so that the drug can be applied, localizedand removed easily.

Administration can be provided by the subject or by another person,e.g., a another caregiver. A caregiver can be any entity involved withproviding care to the human: for example, a hospital, hospice, doctor'soffice, outpatient clinic; a healthcare worker such as a doctor, nurse,or other practitioner; or a spouse or guardian, such as a parent. Themedication can be provided in measured doses or in a dispenser whichdelivers a metered dose.

The subject can also be monitored for an improvement or stabilization ofdisease symptoms.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

In one embodiment, unit doses or measured doses of a composition thatinclude iRNA are dispensed by an implanted device. The device caninclude a sensor that monitors a parameter within a subject. Forexample, the device can include a pump, such as an osmotic pump and,optionally, associated electronics.

An iRNA agent can be packaged in a viral natural capsid or in achemically or enzymatically produced artificial capsid or structurederived therefrom.

Dosage.

An iRNA agent can be administered at a unit dose less than about 75 mgper kg of bodyweight, or less than about 70, 60, 50, 40, 30, 20, 10, 5,2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg ofbodyweight, and less than 200 nmole of RNA agent (e.g., about 4.4×1016copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15,7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015nmole of RNA agent per kg of bodyweight. The unit dose, for example, canbe administered by injection (e.g., intravenous or intramuscular,intrathecally, or directly into an organ), an inhaled dose, or a topicalapplication.

Delivery of an iRNA agent directly to an organ (e.g., directly to theliver) can be at a dosage on the order of about 0.00001 mg to about 3 mgper organ, or preferably about 0.0001-0.001 mg per organ, about 0.03-3.0mg per organ, about 0.1-3.0 mg per eye or about 0.3-3.0 mg per organ.

The dosage can be an amount effective to treat or prevent a disease ordisorder.

In one embodiment, the unit dose is administered less frequently thanonce a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into an siRNA agent, or a DNA which encodes aniRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof). The maintenance dose or doses are generally lowerthan the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 75 mg/kg of body weight per day, e.g., 70,60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or0.0005 mg per kg of bodyweight per day. The maintenance doses arepreferably administered no more than once every 5, 10, or 30 days.Further, the treatment regimen may last for a period of time which willvary depending upon the nature of the particular disease, its severityand the overall condition of the patient. In preferred embodiments thedosage may be delivered no more than once per day, e.g., no more thanonce per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8days. Following treatment, the patient can be monitored for changes inhis condition and for alleviation of the symptoms of the disease state.The dosage of the compound may either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the diseasestate is observed, if the disease state has been ablated, or ifundesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In one embodiment, the iRNA agent pharmaceutical composition includes aplurality of iRNA agent species. The iRNA agent species can havesequences that are non-overlapping and non-adjacent with respect to anaturally occurring target sequence, e.g., a target sequence of the ApoBgene. In another embodiment, the plurality of iRNA agent species isspecific for different naturally occurring target genes. For example, aniRNA agent that targets ApoB can be present in the same pharmaceuticalcomposition as an iRNA agent that targets a different gene. In anotherembodiment, the iRNA agents are specific for different alleles.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g. nasal, buccal, orpulmonary. For example, nasal formulations tend to require much lowerconcentrations of some ingredients in order to avoid irritation orburning of the nasal passages. It is sometimes desirable to dilute anoral formulation up to 10-100 times in order to provide a suitable nasalformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent (e.g., a precursor, e.g., alarger iRNA agent which can be processed into an siRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, orsiRNA agent, or precursor thereof) can include a single treatment or,preferably, can include a series of treatments. It will also beappreciated that the effective dosage of an iRNA agent such as an siRNAagent used for treatment may increase or decrease over the course of aparticular treatment. Changes in dosage may result and become apparentfrom the results of diagnostic assays as described herein. For example,the subject can be monitored after administering an iRNA agentcomposition. Based on information from the monitoring, an additionalamount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g., a gene thatproduces a target ApoB RNA. The transgenic animal can be deficient forthe corresponding endogenous RNA. In another embodiment, the compositionfor testing includes an iRNA agent that is complementary, at least in aninternal region, to a sequence that is conserved between the target ApoBRNA in the animal model and the target ApoB RNA in a human.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1 siRNAs were Produced by Solid-Phase Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

sIRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Glen Research, Sterling Va.) as solid support. RNA and RNAcontaining 2′-O-methyl nucleotides were generated by solid phasesynthesis employing the corresponding phosphoramidites and 2′-O-methylphosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg,Germany). These building blocks were incorporated at selected siteswithin the sequence of the oligoribonucleotide chain using standardnucleoside phosphoramidite chemistry such as described in Currentprotocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.),John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkageswere introduced by replacement of the iodine oxidizer solution with asolution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification by anion exchange HPLC of the crudeoligoribonucleotides were carried out according to establishedprocedures. Yields and concentrations were determined by UV absorptionof a solution of the respective RNA at a wavelength of 260 nm using aspectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim,Germany). Double stranded RNA was generated by mixing an equimolarsolution of complementary strands in annealing buffer (20 mM sodiumphosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at85-90° C. for 3 minutes and cooled to room temperature over a period of3-4 hours. The purified RNA solution was stored at −20° C. until use.

Cholesterol was conjugated to siRNA as illustrated in FIG. 1. For thesynthesis of these 3′-cholesterol-conjugated siRNAs, an appropriatelymodified solid support was used for RNA synthesis. The modified solidsupport was prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole)was added and the mixture was stirred at room temperature until thecompletion of reaction was ascertained by TLC (19 h). After 19 h whichit was partitioned with dichloromethane (3×100 mL). The organic layerwas dried with anhydrous sodium sulfate, filtered and evaporated. Theresidue was distilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It wasthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution was brought to room temperature and stirred further for 6 h.the completion of the reaction was ascertained by TLC. The reactionmixture was concentrated in vacuum and to the ethylacetate was added toprecipitate diisopropyl urea. The suspension was filtered. The filtratewas washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate andwater. The combined organic layer was dried over sodium sulfate andconcentrated to give the crude product which was purified by columnchromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidinein dimethylformamide at 0° C. The solution was continued stirring for 1h. The reaction mixture was concentrated in vacuum and the residue waterwas added and the product was extracted with ethyl acetate. The crudeproduct was purified by converting into hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. Thesuspension was cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) wasadded. The reaction mixture was stirred overnight. The reaction mixturewas diluted with dichloromethane and washed with 10% hydrochloric acid.The product was purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of drytoluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD was added slowly with stirring within 20 mins. Thetemperature was kept below 5° C. during the addition. The stirring wascontinued for 30 mins at 0° C. and 1 mL of glacial acetic acid wasadded, immediately followed by 4 g of NaH₂PO₄.H₂O in 40 mL of water Theresultant mixture was extracted twice with 100 mL of dichloromethaneeach and the combined organic extracts were washed twice with 10 mL ofphosphate buffer each, dried, and evaporated to dryness. The residue wasdissolved in 60 mL of toluene, cooled to 0° C. and extracted with three50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extractswere adjusted to pH 3 with phosphoric acid, and extracted with five 40mL portions of chloroform which were combined, dried and evaporated to aresidue. The residue was purified by column chromatography using 25%ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) was added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) was added, the mixture was extracted with ethylacetate (3×40mL). The combined ethylacetate layer was dried over anhydrous sodiumsulfate and concentrated in vacuum to yield the product which waspurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added withstirring. The reaction was carried out at room temperature overnight.The reaction was quenched by the addition of methanol. The reactionmixture was concentrated in vacuum and to the residue dichloromethane(50 mL) was added. The organic layer was washed with 1M aqueous sodiumbicarbonate. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated. The residual pyridine was removed byevaporating with toluene. The crude product was purified by columnchromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g,95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)esterAH

Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture was dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and thesolution was stirred at room temperature under argon atmosphere for 16h. It was then diluted with dichloromethane (40 mL) and washed with icecold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). Theorganic phase was dried over anhydrous sodium sulfate and concentratedto dryness. The residue was used as such for the next step.

Cholesterol Derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) was added. The reaction mixture turned brightorange in color. The solution was agitated briefly using wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mm/g)was added. The suspension was agitated for 2 h. The CPG was filteredthrough a sintered funnel and washed with acetonitrile, dichloromethaneand ether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The loading capacity of the CPG was measured bytaking UV measurement (37 mM/g).

The synthesis and structure of cholesterol conjugated RNA strands isillustrated in FIG. 1.

Example 2 siRNAs were Designed to Target Regions in Human and Mouse ApoBGenes

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 2.

TABLE 2 Abbreviations of nucleotide monomersused in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation^(a) Nucleotide(s) A, a2′-deoxy-adenosine-5′-phosphate, adenosine-5′- phosphate C, c2′-deoxy-cytidine-5′-phosphate, cytidine-5′-phosphate G, g2′-deoxy-guanosine-5′-phosphate, guanosine-5′- phosphate T, t2′-deoxy-thymidine-5′-phosphate, thymidine-5′- phosphate U, u2′-deoxy-uridine-5′-phosphate, uridine-5′-phosphate Y, y pyrimidine (Cor T, c or u) R, r purine (A or G, a or g) N, n any (G, A, C, or T, g,a, c or u) am 2′-O-methyladenosine-5′-phosphate cm2′-O-methylcytidine-5′-phosphate gm 2′-O-methylguanosine-5′-phosphate tm2′-O-methyl-thymidine-5′-phosphate um 2′-O-methyluridine-5′-phosphate af2′-fluoro-2′-deoxy-adenosine-5′-phosphate cf2′-fluoro-2′-deoxy-cytidine-5′-phosphate gf2′-fluoro-2′-deoxy-guanosine-5′-phosphate tf2′-fluoro-2′-deoxy-thymidine-5′-phosphate uf2′-fluoro-2′-deoxy-uridine-5′-phosphate A, C, G, T, U, a, underlined:nucleoside-5′-phosphorothioate c, g, t, u am, cm, gm, tm, underlined:2-O-methyl-nucleoside-5′-phosphorothioate um

bold italic: 2′-deoxy-adenosine, 2′-deoxy-cytidine,

2′-deoxy-guanosine, 2′-deoxy-thymidine, 2′-deoxy- uridine, adenosine,cytidine, guanosine, thymidine, uridine (5′-hydroxyl)

bold italic: 2′-O-methyl-adenosine, 2′-O-methyl-

cytidine, 2′-O-methyl-guanosine, 2′-O-methyl- thymidine,2′-O-methyl-uridine (5′-hydroxyl) ^(a)capital letters represent2′-deoxyribonucleotides (DNA), lower case letters representribonucleotides (RNA)

Certain oligonucleotides described herein were modified to include acholesterol moiety linked to their 3′-end (see FIG. 1). These aredenoted as 5′-(n)_(n)(Chol)-3′.

Where this text refers to “position n” (n being an integer number)within a given nucleotide sequence, this is meant to refer to the n-thnucleotide in the nucleotide sequence, wherein the 5′-most nucleotide iscounted as the first nucleotide, and counting is continued in the3′-direction.

Since therapeutics for use in humans are typically first tested inanimals, we designed siRNAs that would potentially have an effect bothin an animal model system as well as in a human. The animal model systemchosen was the mouse, mus musculus. Therefore, the first criterion inchoosing sequences for siRNA targeting was cross-reactivity betweenmouse and human ApoB.

In order to select siRNAs that would potentially inhibit ApoB geneexpression in mouse as well as in humans, the sequences coding for theopen reading frame of mouse (GenBank Accession number: XM_(—)137955) andhuman (GenBank Accession number: NM_(—)000384) ApoB were aligned using apair wise BLAST algorithm. The mathematical algorithm used in BLASTprograms is described in Altschul et al. (Nucleic Acids Res.25:3389-3402, 1997). Regions with identity in 23 or more consecutivenucleotides (nucleotides in mouse open reading frame: 463-494, 622-647,658-680, 701-725, 1216-1240, 1282-1320, 1299-1328, 1339-1362, 2124-2155,2807-2830, 2809-2837, 2860-2901, 3035-3057, 3103-3125, 3444-3467,3608-3635, 4130-4167, 4374-4402, 4503-4525, 5962-5985, 6696-6724,9232-9257, 9349-9372, 10177-10213, 10477-10505, 10791-10814,11020-11045, 12227-12251, 13539-13572) were identified.

All possible nucleotide sequences of 23 nucleotides in length weredetermined. This set represented 170 potential siRNA targeting regions.These sequences were compared by BLAST searching (word size 7, mismatchpenalty −1, expect value 1000) against human and mouse genome and mRNAdatabases. All potential 23 nucleotide target regions with 3 or lesssequence mismatches to any non-ApoB sequence in the mouse mRNA and mousegenome databases were excluded from the initial set. The remaining 84potential targeting regions served as template to derive the siRNA senseand antisense strands. The sense strand of each siRNA was identical tonucleotides 3 to 23 (5′ to 3′) from the potential target region. Theantisense strand was defined as the reverse complement of the full 23nucleotide target region. The resulting siRNAs had 2 nucleotideoverhangs at the 3′-end of the antisense strand and a base-paired regionof 21 nucleotides. 81 of these 84 potential siRNAs were synthesized andtheir efficacy in inhibiting the expression of ApoB in cultured humanHepG2 cells was determined. For those siRNAs effecting a repression ofApoB mRNA expression to less than 50% of ApoB mRNA levels in untreatedcontrol cells, the stability in human and/or mouse serum was alsodetermined.

The sequences of the sense and antisense strands of the 84 synthesizedsiRNA duplexes are shown in Table 3. Sense strands represent nucleotides3-23 of all 23 nucleotide regions which are: (a) homologous between theopen reading frames (ORF) of mouse (GenBank Accession number:XM_(—)137955) and human (GenBank Accession number: NM_(—)000384) ApoB;and (b) were found to have 4 or more mismatches when compared to allother entries in the human and mouse genome and mRNA databases. Theantisense strands shown in Table 3 are complementary to nucleotides 1-23of the 23 sense strand nucleotide regions. The position of the 5′-mostnucleotide of the corresponding 23 nucleotide region in the ORF of mouseApoB is also given.

TABLE 3 Nucleic acid sequences of unmodified siRNA duplexes SEQ. SEQ. IDID Sequence antisense Duplex Start No. Sequence sense strand No. stranddescriptor^(a) pos.^(b) 1

agccuugguucaguguggac 2

uccacacugaaccaaggcuugu AL-DUP 5000 1302 3

gaacaccaacuucuuccacg 4

guggaagaaguugguguucauc AL-DUP 5001 2865 5

auugauugaccuguccauuc 6

aauggacaggucaaucaaucuu AL-DUP 5002 13539 7

auggacucaucugcuacagc 8

cuguagcagaugaguccauuug AL-DUP 5003 3610 9

uugaccuguccauucaaaac 10

uuuugaauggacaggucaauca AL-DUP 5004 13544 11

uugugacaaauaugggcauc 12

augcccauauuugucacaaacu AL-DUP 5005 2810 13

uugguucaguguggacagcc 14

gcuguccacacugaaccaaggc AL-DUP 5006 1306 15

ggacucaucugcuacagcuu 16

agcuguagcagaugaguccauu AL-DUP 5007 3612 17

uugauugaccuguccauuca 18

gaauggacaggucaaucaaucu AL-DUP 5008 13540 19

ugauugaccuguccauucaa 20

ugaauggacaggucaaucaauc AL-DUP 5009 13541 21

aaauggacucaucugcuaca 22

guagcagaugaguccauuugga AL-DUP 5010 3608 23

auugaccuguccauucaaaa 24

uuugaauggacaggucaaucaa AL-DUP 5011 13543 25

gauugaccuguccauucaaa 26

uugaauggacaggucaaucaau AL-DUP 5012 13542 27

guguauggcuucaacccuga 28

caggguugaagccauacaccuc AL-DUP 5013 466 29

cugugggauuccaucugcca 30

ggcagauggaaucccacagacu AL-DUP 5014 4136 31

gacuuccugaauaacuaugc 32

cauaguuauucaggaagucuau AL-DUP 5015 9349 33

caauuugaucaguauauuaa 34

uaauauacugaucaaauuguau AL-DUP 5016 6697 35

gacucaucugcuacagcuua 36

aagcuguagcagaugaguccau AL-DUP 5017 3613 37

uacuccaacgccagcuccac 38

uggagcuggcguuggaguaagc AL-DUP 5018 3103 39

ugacaaauaugggcaucauc 40

augaugcccauauuugucacaa AL-DUP 5019 2813 41

uguauggcuucaacccugag 42

ucaggguugaagccauacaccu AL-DUP 5020 467 43

accguguauggaaacugcuc 44

agcaguuuccauacacgguauc AL-DUP 5021 703 45

auaccguguauggaaacugc 46

caguuuccauacacgguaucca AL-DUP 5022 701 47

aaucaagugucaucacacug 48

agugugaugacacuugauuuaa AL-DUP 5023 10178 49

gguguauggcuucaacccug 50

aggguugaagccauacaccucu AL-DUP 5024 465 51

uuugugacaaauaugggcau 52

ugcccauauuugucacaaacuc AL-DUP 5025 2809 53

uaccguguauggaaacugcu 54

gcaguuuccauacacgguaucc AL-DUP 5026 702 55

aaaucaagugucaucacacu 56

gugugaugacacuugauuuaaa AL-DUP 5027 10177 57

agguguauggcuucaacccu 58

ggguugaagccauacaccucuu AL-DUP 5028 464 59

ggcuucaacccugagggcaa 60

ugcccucaggguugaagccaua AL-DUP 5029 472 61

aacaccaacuucuuccacga 62

cguggaagaaguugguguucau AL-DUP 5030 2866 63

uauggcuucaacccugaggg 64

ccucaggguugaagccauacac AL-DUP 5031 469 65

uggcuucaacccugagggca 66

gcccucaggguugaagccauac AL-DUP 5032 471 67

acaccaacuucuuccacgag 68

ucguggaagaaguugguguuca AL-DUP 5033 2867 69

caccaacuucuuccacgagu 70

cucguggaagaaguugguguuc AL-DUP 5034 2868 71

accaacuucuuccacgaguc 72

acucguggaagaaguugguguu AL-DUP 5035 2869 73

augaacaccaacuucuucca 74

ggaagaaguugguguucaucug AL-DUP 5036 2863 75

ugaacaccaacuucuuccac 76

uggaagaaguugguguucaucu AL-DUP 5037 2864 77

gaugaacaccaacuucuucc 78

gaagaaguugguguucaucugg AL-DUP 5038 2862 79

uuccaucugccaucucgaga 80

cucgagauggcagauggaaucc AL-DUP 5039 4144 81

uccaucugccaucucgagag 82

ucucgagauggcagauggaauc AL-DUP 5040 4145 83

caagccuugguucagugugg 84

cacacugaaccaaggcuuguaa AL-DUP 5041 1300 85

ucaagucugugggauuccau 86

uggaaucccacagacuugaggu AL-DUP 5042 4130 87

aucaagugucaucacacuga 88

cagugugaugacacuugauuua AL-DUP 5043 10179 89

auggcuucaacccugagggc 90

cccucaggguugaagccauaca AL-DUP 5044 470 91

ugaccuguccauucaaaacu 92

guuuugaauggacaggucaauc AL-DUP 5045 13545 93

ucaagugucaucacacugaa 94

ucagugugaugacacuugauuu AL-DUP 5046 10180 95

caagugucaucacacugaau 96

uucagugugaugacacuugauu AL-DUP 5047 10181 97

ucaucacacugaauaccaau 98

uugguauucagugugaugacac AL-DUP 5048 10187 99

uguccauucaaaacuaccac 100

ugguaguuuugaauggacaggu AL-DUP 5049 13550 101

cuguccauucaaaacuacca 102

gguaguuuugaauggacagguc AL-DUP 5050 13549 103

ucacacugaauaccaaugcu 104

gcauugguauucagugugauga AL-DUP 5051 10190 105

ccuguccauucaaaacuacc 106

guaguuuugaauggacagguca AL-DUP 5052 13548 107

accuguccauucaaaacuac 108

uaguuuugaauggacaggucaa AL-DUP 5053 13547 109

aucacacugaauaccaaugc 110

cauugguauucagugugaugac AL-DUP 5054 10189 111

acaagccuugguucagugug 112

acacugaaccaaggcuuguaaa AL-DUP 5055 1299 113

guauggcuucaacccugagg 114

cucaggguugaagccauacacc AL-DUP 5056 468 115

gaccuguccauucaaaacua 116

aguuuugaauggacaggucaau AL-DUP 5057 13546 117

caucacacugaauaccaaug 118

auugguauucagugugaugaca AL-DUP 5058 10188 119

ugugacaaauaugggcauca 120

gaugcccauauuugucacaaac AL-DUP 5059 2811 121

aagugucaucacacugaaua 122

auucagugugaugacacuugau AL-DUP 5060 10182 123

aacacuaagaaccagaagau 124

ucuucugguucuuaguguuagc AL-DUP 5061 11020 125

aauuugaucaguauauuaaa 126

uuaauauacuguacaaauugua AL-DUP 5062 6698 127

ugaacaucaagaggggcauc 128

augccccucuugauguucagga AL-DUP 5084 623 129

gaacaucaagaggggcauca 130

gaugccccucuugauguucagg AL-DUP 5085 624 131

uccagccccaucacuuuaca 132

guaaagugauggggcuggacac AL-DUP 5086 1282 133

agccccaucacuuuacaagc 134

cuuguaaagugauggggcugga AL-DUP 5087 1285 135

gccccaucacuuuacaagcc 136

gcuuguaaagugauggggcugg AL-DUP 5088 1286 137

aguuugugacaaauaugggc 138

cccauauuugucacaaacucca AL-DUP 5089 2807 139

gggaaucuuauauuugaucc 140

gaucaaauauaagauucccuuc AL-DUP 5090 2131 141

uacugagcugagaggccuca 142

gaggccucucagcucaguaacc AL-DUP 5091 1218 143

uugggaagaagaggcagcuu 144

agcugccucuucuucccaauua AL-DUP 5092 12228 145

cacauccuccaguggcugaa 146

ucagccacuggaggaugugagu AL-DUP 5093 1339 147

ccccaucacuuuacaagccu 148

ggcuuguaaagugauggggcug AL-DUP 5094 1287 149

cagccccaucacuuuacaag 150

uuguaaagugauggggcuggac AL-DUP 5095 1284 151

agggaaucuuauauuugauc 152

aucaaauauaagauucccuucu AL-DUP 5096 2130 153

uuuacaagccuugguucagu 154

cugaaccaaggcuuguaaagug AL-DUP 5097 1296 155

gaaucuuauauuugauccaa 156

uggaucaaauauaagauucccu AL-DUP 5098 2133 157

aagggaaucuuauauuugau 158

ucaaauauaagauucccuucua AL-DUP 5099 2129 159

aauagaagggaaucuuauau 160

uauaagauucccuucuauuuug AL-DUP 5100 2124 161

agaagggaaucuuauauuug 162

aaauauaagauucccuucuauu AL-DUP 5101 2127 163 gacuuccugaauaacuaugca 164ugcauaguuauucaggaagucua 9350 165 gcaaggaucuggagaaacaac 166guuguuucuccagauccuugcac 4375 167 caaggaucuggagaaacaaca 168uguuguuucuccagauccuugca 4376 ^(a)Descriptor refers to annealed duplexsiRNA ^(b)Position of the 5′-most nucleotide of th e corresponding 23nucleotide region in the OFR of mouse ApoB

Alternatively, for the same set of 84 potential target regions, siRNAsmay be generated with 19 basepairs and 2 nucleotide dTdT overhangs. Thesense strand is then identical to nucleotides 1 to 19, 2 to 20, 3 to 21,4 to 22, or 5 to 23 from the target region, and two dT nucleotides areadded to the 3″-end of the oligonucleotide. The reverse complement ofthe sense strand so selected would then serve as template for theantisense strand, and two dT nucleotides would be added to the 3″-end.

Example 3 siRNAs Inhibited ApoB Expression Both on the mRNA as Well asthe Protein Level, in Cell Culture

The activity of the siRNAs described above was tested in HepG2 cells.

HepG2 cells in culture were used for quantitation of ApoB mRNA in totalmRNA isolated from cells incubated with ApoB-specific siRNAs by branchedDNA assay, and of ApoB 100 protein in supernatant of cells incubatedwith ApoB-specific siRNAs by Enzyme-linked immunosorbent assay (ELISA).HepG2 cells were obtained from American Type Culture Collection(Rockville, Md., cat. No. HB-8065) and cultured in MEM (GibcoInvitrogen, Invitrogen GmbH, Karlsruhe, Germany, cat. No. 21090-022)supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin,Germany, cat. No. S0115), 2 mM L-Glutamin (Biochrom AG, Berlin, Germany,cat. No. K0238), Penicillin 100 U/ml, Streptomycin 100 μg/ml (BiochromAG, Berlin, Germany, cat. No. A2213), 1× non-essential amino acids (NEA)(Biochrom AG, Berlin, Germany, cat. No. K0293) and 1 mM sodium pyruvate(Biochrom AG, Berlin, Germany, cat. No. L0473) at 37° C. in anatmosphere with 5% CO₂ in a humidified incubator (Heraeus HERAcell,Kendro Laboratory Products, Langenselbold, Germany).

For transfection with siRNA, HepG2 cells were seeded at a density of1.5×10⁴ cells/well in 96-well plates and cultured for 24 hours.Transfection of siRNA was carried out with oligofectamine (InvitrogenGmbH, Karlsruhe, Germany, cat. No. 12252-011) as described by themanufacturer. SiRNAs were transfected at a concentration of 100 nM forthe screening of siRNA duplexes, and 100, 33, 11, 3.7, 1.2, 0.4, 0.14,and 0.05 nM when assessing dose response/inhibitor concentration at 50%maximal inhibition (IC₅₀). 24 hours after transfection, the medium waschanged and cells were incubated for an additional 24 hours. For theassessment of ApoB100 protein concentration by enzyme-linkedimmunosorbent assay, as described below, supernatant was collected andstored at −80° C. until analysis. For measurement of ApoB mRNA bybranched DNA assay, as described below, cells were harvested and lysedfollowing procedures recommended by the manufacturer of the QuantigeneExplore Kit (Genospectra, Fremont, Calif., USA, cat. No. QG-000-02) forbDNA quantitation of mRNA, except that 2 μl of a 50 μg/μl stock solutionof Proteinase K (Epicentre, Madison, Wis., USA, Cat. No. MPRK092) wasadded to 600 μl of Tissue and Cell Lysis Solution (Epicentre, Madison,Wis., USA, cat. No. MTC096H). Lysates were stored at −80° C. untilanalysis by branched DNA assay.

NmuLi cells in culture were used for quantitation of murine ApoB mRNA bybranched DNA assay (bDNA assay). NmuLi cells (normal murine liver, ATCCNumber: CRL-1638) were cultured in DMEM (Biochrom AG, Berlin, Germany,cat. No. F0435) supplemented to contain 10% fetal calf serum (FCS)(Biochrom AG, Berlin, Germany, cat. No. 50115) and 2 mM L-Glutamin(Biochrom AG, Berlin, Germany, cat. No. K0238) at 37° C. under anatmosphere containing 5% CO₂ in a humidified incubator (HeraeusHERAcell, Kendro Laboratory Products, Langenselbold, Germany).

One day before transfection, 4×10³ cells per well were seeded on 96-wellplates. Cells were transfected with siRNAs in triplicate witholigofectamine according to the manufacturer's protocol (InvitrogenGmbH, Karlsruhe, Germany, cat. No. 12252-011). Concentration of thesiRNA in the medium during transfection was 200 nM for the screening of15 unmodified or modified siRNA duplexes. Following transfection, cellswere cultured for 24 h, after which the growth medium was exchanged forfresh medium not containing the siRNA. Cell lysates were obtained andstored as described above for HepG2 cells.

The sequences of an siRNA duplex used as a non-cholesterol conjugatedcontrol is shown below:

AL-DUP HCV SEQ. ID No. 169 Sense: 5′-

cggcuagcugugaaaggucc-3′ SEQ. ID No. 170 Antisense: 5′-

gaccuuucacagcuagccguga-3′

The sense strand of AL-DUP HCV corresponds to positions 9472-9493 of the3′-untranslated region of hepatitis C virus (Accession number: D89815).

The sequences of an siRNA duplex used as a cholesterol-conjugatedcontrol is shown below:

AL-DUP 5129 SEQ. ID No. 171 Sense: 5′-ccacaugaagcagcacgacuu(Chol)-3′SEQ. ID No. 172 Antisense: 5′-aagucgugcugcuucaugug-3′

Nucleotides 1-21 of the sense strand correspond to positions 843-864 incloning vector pEGFP-C3 with enhanced green fluorescent protein (GenBankAccession number: U57607).

ApoB100 protein levels in cell supernatants were measured by ELISAassay. Clear Flat Bottom Polystyrene High Bind Microplates (Corning B.V.Life Sciences, Schiphol-Rijk, The Netherlands, cat. no. 9018) were usedfor the assays. Polyclonal antibody goat anti-human-apolipoprotein B(Chemicon International GmbH, Hofheim, Germany, cat. no. AB742) wasdiluted 1:1000 in phosphate buffered saline (PBS) (PBS Dulbecco w/oCa²⁺, Mg²⁺, Biochrom AG, Berlin, Germany, cat. No. L182-05) and 100 μlof this dilution was coated on 96-well plates at 4° C. overnight. Afterblocking with 300 μl of 1% bovine serum albumin (BSA) (Carl Roth GmbH &Co KG, Karlsruhe, Germany, cat. no. 8076.2) in PBS the plate was washedthree times with PBS.

Cell culture supernatant was thawed and diluted 1:1 with PBS containing0.1% Tween 20 (Carl Roth GmbH & Co KG, Karlsruhe, Germany, cat. No.9127.1) and 0.1% BSA. 100 μl of this dilution was added to each well.After an incubation time of 2 hours at room temperature, the plate waswashed five times with PBS containing 0.1% Tween 20 followed by threewashes with PBS. 100 μl of a horseradish-peroxidase conjugated GoatAnti-Human Apolipoprotein B-100 polyclonal antibody (Academy Bio-MedicalCompany, Houston, Tex., USA, cat. No. 20H-G1-b) diluted 1:1000 in PBScontaining 0.1% Tween 20 and 3% BSA was added to each well. The platewas incubated for 2 hours at room temperature. After washing the platefive times with PBS containing 0.1% Tween 20 and three times with PBS,wells were incubated with 0.9 mg/ml OPD (o-phenylendiaminedihydrochloride, Merck Biosciences GmbH, Bad Soden, Germany cat. No.523121) in 24 mmol/L citric acid buffer (Sigma-Aldrich, Taufkirchen,Germany, cat. no. C1909-1KG), pH 5.0, containing 0.03% hydrogen peroxide(Merck Biosciences GmbH, Bad Soden, Germany cat. No. 386790). The enzymereaction was halted by adding 0.5 mol/L H₂SO₄ (Merck KgaA, Darmstadt,Germany, cat. No. 100731) and absorbance at 490 nm was measured on aspectrophotometer (Perkin Elmer Wallac Victor3 1420 multilabel reader,PerkinElmer LAS GmbH, Rodgau, Germany). siRNA duplexes unrelated to anymouse gene were used as control, and the activity of a given ApoBspecific siRNA duplex was expressed as percent ApoB proteinconcentration in the supernatant of treated cells relative to ApoBprotein concentration in the supernatant of cells treated with thecontrol siRNA duplex. The conjugation of a cholesterol moiety to thesense strand of siRNA duplexes enhanced the ApoB secretion-inducingeffect in cultured HepG2 cells. Therefore, one siRNA duplex controlincluded a conjugated cholesterol moiety (AL-DUP 5129).

ApoB100 mRNA levels were measured by branched-DNA (bDNA) assay. Theassay was performed using the Quantigene Explore Kit (Genospectra,Fremont, Calif., USA, cat. No. QG-000-02). Frozen lysates were thawed atroom temperature, and ApoB and GAPDH mRNA quantified using theQuantigene Explore Kit according to manufacturer's instructions. Nucleicacid sequences for Capture Extender (CE), Label Extender (LE) andblocking (BL) probes were selected from the nucleic acid sequences ofApoB and GAPDH with the help of the QuantiGene ProbeDesigner Software2.0 (Genospectra, Fremont, Calif., USA, cat. No. QG-002-02). Probenucleotide sequences used in quantization of murine and human ApoB areshown in Table 4 and Table 5, respectively. Probe nucleotide sequencesused in quantization of murine and human GAPDH are shown in Table 6 andTable 7, respectively.

TABLE 4 DNA probes for murine ApoB used in branched-DNA assays SEQ. ID.Probe type^(a) Nucleotide sequence No. CE

TCATTCTCCAGCAGCAGGGTTTTTCTCTTGGAAAGAAAGT 173 CE

AAGCGGCCGTTTGTTGATATTTTTCTCTTGGAAAGAAAGT 174 CE

TTTTTGCTGTCTGCACCCATTTTTCTCTTGGAAAGAAAGT 175 CE

AAATATTGTCCATTTTTGAGAAGAAGTTTTTCTCTTGGAAAGAAAGT 176 CE

ATTCAGCTTCAGTGGCTCCATTTTTCTCTTGGAAAGAAAGT 177 CE

ATGTCTGCATTTAGCCTATGGCTTTTTTCTCTTGGAAAGAAAGT 178 LE

GCCCAAGCTCTGCATTCAATTTTTAGGCATAGGACCCGTGTCT 179 LE

TTTCATGGATGCCCCAGAGTTTTTAGGCATAGGACCCGTGTCT 180 LE

CTGAATTTTGCATGGTGTTCTTTTTTTTAGGCATAGGACCCGTGTCT 181 LE

GGCAGCTCTCCCATCAAGTTTTTAGGCATAGGACCCGTGTCT 182 LE

AATCATGGCCTGGTAAATGCTTTTTAGGCATAGGACCCGTGTCT 183 LE

AGCATAGGAGCCCATCAAATCATTTTTTAGGCATAGGACCCGTGTCT 184 LE

ACTGTGTGTGTGGTCAAGTTTCATCTTTTTTAGGCATAGGACCCGTGTCT 185 LE

TAGGGCTGTAGCTGTAAGTTAAAATTTTTTAGGCATAGGACCCGTGTCT 186 LE

TCAAATCTAGAGCACCATATCTCAGTTTTTAGGCATAGGACCCGTGTCT 187 LE

CCGAAACCTTCCATTGTTGTTTTTAGGCATAGGACCCGTGTCT 188 LE

GATATGTTTCAGCTCATTATTTTGATAGTTTTTAGGCATAGGACCCGTGTCT 189 LE

TACTACCAGGTCAGTATAAGATATGGTATTTTTTAGGCATAGGACCCGTGTCT 190 LE

AATTCGACACCCTGAACCTTAGTTTTTAGGCATAGGACCCGTGTCT 191 BL

CCCCAGTGACACCTCTGTGA 192 BL

CGGCTGAGTTTGAAGTTGAAGAT 193 BL

GGACAGCCTCAGCCCTTC 194 BL

CCAGTGAGAGACCTGCAATGTTCA 195 BL

CTGCTTATAGAACTTGTCTCCACTG 196 BL

TCGTTGCTTAAAGTAGTTATGAAAGA 197 BL

TTCCTTTAAAGTTGCCACCCA 198 BL

CACAGTGTCTGCTCTGTAACTTG 199 ^(a)CE = Capture Extender probe; LE = LabelExtender probe; BL = blocking probe

TABLE 5 DNA probes for human ApoB used in branched-DNA assaysProbe type^(a) Nucleotide sequence SEQ. ID. No. CE

ATTGGATTTTCAGAATACTGTATAGCTTTTTTCTCTTGGAAAGAAAGT 200 CE

CTGCTTCGTTTGCTGAGGTTTTTTCTCTTGGAAAGAAAGT 201 CE

CAGTGATGGAAGCTGCGATATTTTTCTCTTGGAAAGAAAGT 202 CE

AACTTCTAATTTGGACTCTCCTTTGTTTTTCTCTTGGAAAGAAAGT 203 CE

CTCCTTCAGAGCCAGCGGTTTTTCTCTTGGAAAGAAAGT 204 CE

CTCCCATGCTCCGTTCTCATTTTTCTCTTGGAAAGAAAGT 205 CE

GGGTAAGCTGATTGTTTATCTTGATTTTTCTCTTGGAAAGAAAGT 206 LE

GTTCCATTCCCTATGTCAGCATTTTTAGGCATAGGACCCGTGTCT 207 LE

TTAATCTTAGGGTTTGAGAGTTGTGTTTTTAGGCATAGGACCCGTGTCT 208 LE

ACTGTGTTTGATTTTCCCTCAATATTTTTAGGCATAGGACCCGTGTCT 209 LE

GTATTTTTTTCTGTGTGTAAACTTGCTTTTTAGGCATAGGACCCGTGTCT 210 LE

AATCACTCCATTACTAAGCTCCAGTTTTTAGGCATAGGACCCGTGTCT 211 BL

GCCAAAAGTAGGTACTTCAATTG 212 BL

TTGCATCTAATGTGAAAAGAGGA 213 BL

ATTTGCTTGAAAATCAAAATTGA 214 BL

GTACTTGCTGGAGAACTTCACTG 215 BL

CATTTCCAAAAAACAGCATTTC 216 ^(a)CE = Capture Extender probe; LE = LabelExtender probe; BL = blocking probe

TABLE 6 DNA probes for murine GAPDH used in branched-DNA assaysProbe type^(a) Nucleotide sequence SEQ. ID. No. CE

AAATGGCAGCCCTGGTGATTTTTCTCTTGGAAAGAAAGT 217 CE

CTTGACTGTGCCGTTGAATTTTTTTTCTCTTGGAAAGAAAGT 218 CE

TCTCGCTCCTGGAAGATGGTTTTTCTCTTGGAAAGAAAGT 219 CE

CCGGCCTTCTCCATGGTTTTTTCTCTTGGAAAGAAAGT 220 LE

ACAATCTCCACTTTGCCACTGTTTTTAGGCATAGGACCCGTGTCT 221 LE

ATGTAGACCATGTAGTTGAGGTCAATTTTTAGGCATAGGACCCGTGTCT 222 LE

ACAAGCTTCCCATTCTCGGTTTTTAGGCATAGGACCCGTGTCT 223 LE

GATGGGCTTCCCGTTGATTTTTTAGGCATAGGACCCGTGTCT 224 LE

ACATACTCAGCACCGGCCTTTTTTAGGCATAGGACCCGTGTCT 225 BL

GAAGGGGTCGTTGATGGC 226 BL

CGTGAGTGGAGTCATACTGGAA 227 BL

ACCCCATTTGATGTTAGTGGG 228 BL

GTGAAGACACCAGTAGACTCCAC 229 ^(a)CE = Capture Extender probe; LE = LabelExtender probe; BL = blocking probe

TABLE 7 DNA probes for human GAPDH used in branched-DNA assaysProbe type^(a) Nucleotide sequence SEQ. ID. No. CE

AATTTGCCATGGGTGGAATTTTTTCTCTTGGAAAGAAAGT 230 CE

GAGGGATCTCGCTCCTGGATTTTTCTCTTGGAAAGAAAGT 231 CE

CCCAGCCTTCTCCATGGTTTTTTCTCTTGGAAAGAAAGT 232 CE

CTCCCCCCTGCAAATGAGTTTTTCTCTTGGAAAGAAAGT 233 LE

GCCTTGACGGTGCCATGTTTTTAGGCATAGGACCCGTGTCT 234 LE

ATGACAAGCTTCCCGTTCTCTTTTTAGGCATAGGACCCGTGTCT 235 LE

GATGGTGATGGGATTTCCATTTTTTTAGGCATAGGACCCGTGTCT 236 LE

CATCGCCCCACTTGATTTTTTTTTAGGCATAGGACCCGTGTCT 237 LE

ACGACGTACTCAGCGCCATTTTTAGGCATAGGACCCGTGTCT 238 LE

GCAGAGATGATGACCCTTTTGTTTTTAGGCATAGGACCCGTGTCT 239 BL

GTGAAGACGCCAGTGGACTC 240 ^(a)CE = Capture Extender probe; LE = LabelExtender probe; BL = blocking probe

The ApoB mRNA levels were normalized across different samples bycomparing the ratio of ApoB mRNA to GAPDH mRNA present in the samples.AL-DUP HCV, which does not target any mouse gene, and the cholesterolconjugated AL-DUP 5129 were used as controls. The activity of a givenApoB specific siRNA duplex was expressed as a percentage of ApoB mRNA(ApoB mRNA/GAPDH mRNA) in treated cells relative to cells treated withthe control siRNA.

Table 8 shows the results from screening 81 siRNA duplexes for theiractivity in reducing ApoB mRNA levels in HepG2 cell cultures and ApoBprotein levels in the supernatant of NmuLi cell cultures.

TABLE 8 Percentage ApoB mRNA and protein following treatment with siRNA(ApoB mRNA/GAPDH ApoB protein in HepG2 mRNA) in HEPG2 cultured cellculture supernatant Duplex descriptor cells relative to controlsrelative to controls AL-DUP 5000 204 35 AL-DUP 5001 141 37 AL-DUP 500268 29 AL-DUP 5003 121 119 AL-DUP 5004 55 55 AL-DUP 5005 250 129 AL-DUP5006 174 99 AL-DUP 5007 96 72 AL-DUP 5008 93 67 AL-DUP 5009 68 92 AL-DUP5010 79 41 AL-DUP 5011 98 44 AL-DUP 5012 111 40 AL-DUP 5013 37 24 AL-DUP5014 112 43 AL-DUP 5015 165 54 AL-DUP 5016 108 44 AL-DUP 5017 117 46AL-DUP 5018 414 93 AL-DUP 5019 46 56 AL-DUP 5020 43 43 AL-DUP 5021 10345 AL-DUP 5022 86 26 AL-DUP 5023 218 74 AL-DUP 5024 25 19 AL-DUP 5025 6447 AL-DUP 5026 84 70 AL-DUP 5027 45 51 AL-DUP 5028 41 31 AL-DUP 5029 4429 AL-DUP 5030 49 27 AL-DUP 5031 45 36 AL-DUP 5032 82 47 AL-DUP 5033 11587 AL-DUP 5034 58 38 AL-DUP 5035 46 26 AL-DUP 5036 47 24 AL-DUP 5037 12053 AL-DUP 5038 62 33 AL-DUP 5039 56 45 AL-DUP 5040 78 70 AL-DUP 5041 38745 AL-DUP 5042 232 52 AL-DUP 5043 65 54 AL-DUP 5044 95 55 AL-DUP 5045 6557 AL-DUP 5046 28 37 AL-DUP 5047 29 56 AL-DUP 5048 28 16 AL-DUP 5049 3136 AL-DUP 5050 55 54 AL-DUP 5051 65 55 AL-DUP 5052 49 49 AL-DUP 5053 3746 AL-DUP 5054 54 43 AL-DUP 5055 205 101 AL-DUP 5056 67 72 AL-DUP 505777 66 AL-DUP 5058 85 37 AL-DUP 5059 116 61 AL-DUP 5060 45 35 AL-DUP 506140 43 AL-DUP 5062 63 47 AL-DUP 5084 26 52 AL-DUP 5085 35 57 AL-DUP 508636 69 AL-DUP 5087 71 27 AL-DUP 5088 35 28 AL-DUP 5089 26 33 AL-DUP 509064 51 AL-DUP 5091 76 90 AL-DUP 5092 37 81 AL-DUP 5093 21 64 AL-DUP 509415 29 AL-DUP 5095 54 57 AL-DUP 5096 55 62 AL-DUP 5097 8 29 AL-DUP 509811 24 AL-DUP 5099 43 48 AL-DUP 5100 17 57 AL-DUP 5101 15 39

The 27 most active siRNA duplexes of Table 8 were determined to be thosewith a residual ApoB mRNA/GAPDH mRNA <31% of controls or residual ApoBprotein levels <35% of controls. These siRNA duplexes were chosen forfurther analysis and establishment of 1050 values. These were: AL-DUP5000, AL-DUP 5002, AL-DUP 5013, AL-DUP 5022, AL-DUP 5024, AL-DUP 5028,AL-DUP 5029, AL-DUP 5030, AL-DUP 5035, AL-DUP 5036, AL-DUP 5038, AL-DUP5046, AL-DUP 5047, AL-DUP 5048, AL-DUP 5049, AL-DUP 5060, AL-DUP 5083,AL-DUP 5084, AL-DUP 5087, AL-DUP 5088, AL-DUP 5089, AL-DUP 5093, AL-DUP5094, AL-DUP 5097, AL-DUP 5098, AL-DUP 5100, AL-DUP 5101.

Dose escalation studies were performed using the above-mentioned 27siRNA duplexes, where ApoB mRNA was quantified in NmuLi cells and ApoBprotein was quantified in cell culture supernatant of HepG2 cells afterincubation with 100, 33, 11, 3.7, 1.2, 0.4, 0.14, or 0.05 nM solutionsof the respective siRNA duplex. The minimum residual ApoB mRNA and ApoBprotein levels were determined. For those 15 of the above 27 siRNAshowing the lowest combined minimum residual ApoB mRNA and proteinlevels, the dose escalation was repeated three times, the resulting datawere used to calculate inhibitor concentration at 50% maximal inhibition(IC50), and an average value was computed over the three determinations.IC50 was calculated by applying the data from the dose escalationexperiments to curve fitting routines implemented in the computersoftware Xlfit 4 (ID Business Solutions Ltd., Guildford, UK). IC50values were computed using the parameterized equations obtained from theline fit using the following parameters: Dose Response One Site, 4Parameter Logistic Model, fit=(A+((B−A)/(1+(((10^C)/x)^D)))),inv=((10^C)/((((B−A)/(y−A))−1)^(1/D))), res=(y−fit) (by way of example,see FIG. 2).

Table 9 shows the average IC50 values for the five ApoB siRNAs thatreduced both mRNA and protein levels by >70% in NmuLi cells. Controlexperiments measured minimal residual ApoB mRNA/GAPDH mRNA in culturedNmuLi cells in percentage of untreated controls, and minimal residualApoB protein in HepG2 cell supernatant in percentage of untestedcontrols

TABLE 9 IC50 of selected siRNAs Minimum Minimum residual residual IC₅₀ApoB mRNA/ ApoB protein in Duplex (ApoB protein GAPDH mRNA cellsupernatant denominator concentration) in % of controls in % of controlsAL-DUP 5097 0.3 nM 15% 18% AL-DUP 5098 0.7 nM 9% 20% AL-DUP 5094 0.7 nM14% 7% AL-DUP 5048 0.9 nM 11% 6% AL-DUP 5024 2.8 nM 12% 21%

AL-DUP 5024 and AL-DUP 5048 were chosen for further investigations.

Example 6 siRNA Duplexes were Modified and Exhibited Improved Resistanceto Nucleases

The siRNA duplexes AL-DUP 5024 and AL-DUP 5048 were altered with variouschemical modifications in an attempt to enhance the resistance of theoligonucleotide strands against degradation by nucleases present inbiological fluids, such as, for example, serum and the intracellularmedium. Specifically, phosphorothioate linkages were introduced betweenpositions 21 and 22 and between positions 22 and 23 of the antisensestrands, and/or between positions 20 and 21 of the sense strands (seeTable 10), thereby increasing the stability of the siRNAs againstexonucleolytic degradation.

TABLE 10 siRNAs for stability assays SEQ. SEQ Duplex ID ID descriptorNo. Sense strand sequence No. .Antisense strand sequenceSiRNA duplexes derived from AL-DUP 5024 AL-DUP 5163 241

gguguauggcuucaacccug(chol) 242

aggguugaagccauacaccumcmu AL-DUP 5164 243

ggumgumaumggcumucmaacccumg(chol) 244

magggumumgaagccmaumacmaccucu AL-DUP 5165 241

gguguauggcuucaacccug(chol) 244

magggumumgaagccmaumacmaccucu AL-DUP 5166 243

ggumgumaumggcumucmaacccumg(chol) 242

aggguugaagccauacaccumcmu AL-DUP 5180 249

ggumgumaumggcumucmaacccumg 242

aggguugaagccauacaccumcmu AL-DUP 5181 249

ggumgumaumggcumucmaacccumg 244

magggumumgaagccmaumacmaccucu SiRNA duplexes derived from AL-DUP 5048AL-DUP 5167 253

ucaucacacugaauaccaau(chol) 254

uugguauucagugugaugacmamc AL-DUP 5168 255

ucmaucmacmacumgaaumaccmaau(chol) 256

umumggumaumucmagumgumgaumgacmac AL-DUP 5169 253

ucaucacacugaauaccaau(chol) 256

umumggumaumucmagumgumgaumgacmac AL-DUP 5170 255

ucmaucmacmacumgaaumaccmaau(chol) 254

uugguauucagugugaugacmamc AL-DUP 5182 261

ucmaucmacmacumgaaumaccmaau 254

uugguauucagugugaugacmamc AL-DUP 5183 261

ucmaucmacmacumgaaumaccmaau 256

umumggumaumucmagumgumgaumgacmac m = 2′O-methyl modification; “(chol)”indicates cholesterol conjugated to the 3′-end via a pyrrolidine linkercomprising a phosphorothioate

Previously, this laboratory identified certain sequence motifs in siRNAduplexes which are particularly prone to degradative attack byendonucleases (see co-owned and co-pending application U.S. 60/574,744).Specifically, these motifs are 5′-UA-3′, 5′-UG-3′, 5′-CA-3′, 5′-UU-3′,and 5′-CC-3′. SiRNAs comprising these sequence motifs can be stabilizedtowards degradative attack by endonucleases by replacing the 2′-OH ofthe ribose subunit of the 5′-most nucleotide in these dinucleotidemotifs with 2′-O—CH₃ (also referred to herein as 2′-O-Methyl or2′-O-Me). Hence, siRNAs were synthesized wherein the respectivenucleotides bear a 2′-O-Me group in all occurrences of thesedinucleotide motifs, except for occurrences of 5′-CC-3′, on either thesense strand, the antisense strand, or both.

A further modification tested was the conjugation of a cholesterolmoiety to the 3′-end of the sense strand of the siRNAs (see FIG. 1).This modification is thought to facilitate the uptake of RNA into cells(Manoharan, M. et al., Antisense and Nucleic Acid Drug Development 2002,12:103-128).

The siRNA duplexes listed in Table 10 were synthesized and tested fortheir stability towards nucleolytic degradation in the serum incubationassay, as well as their activity in reducing the amount of ApoB proteinsecreted into supernatant by cultured NmuLi cells.

The nucleotide sequences of siRNA AL-DUP 5024, AL-DUP 5163, AL-DUP 5164,AL-DUP 5165, AL-DUP 5166, AL-DUP 5180, and AL-DUP 5181 are identicalexcept for the following:

AL-DUP 5024 consists entirely of unmodified nucleotides;

AL-DUP 5163 bears 2′-O-Me groups in positions 21 and 22 andphosphorothioate linkages between positions 21 and 22, and 22 and 23, ofthe antisense strand, and a cholesterol moiety conjugated to the 3′-endof the sense strand;

AL-DUP 5164 bears 2′-O-Me groups in positions 4, 6, 8, 12, 14, and 20 ofits sense strand and in positions 1, 6, 7, 13, 15, and 17 of itsantisense strand, phosphorothioate linkages between positions 21 and 22,and 22 and 23, of the antisense strand, and a cholesterol moietyconjugated to the 3′-end of the sense strand;

AL-DUP 5165 bears 2′-O-Me groups in positions 1, 6, 7, 13, 15, and 17 ofits antisense strand, phosphorothioate linkages between positions 21 and22, and 22 and 23, of the antisense strand, and a cholesterol moietyconjugated to the 3′-end of the sense strand;

AL-DUP 5166 bears 2′-O-Me modifications in positions 4, 6, 8, 12, 14,and 20 of its sense strand and in positions 21 and 22 of its antisensestrand, phosphorothioate linkages between positions 21 and 22, and 22and 23, of the antisense strand, and a cholesterol moiety conjugated tothe 3′-end of the sense strand;

AL-DUP 5180 bears 2′-O-Me modifications in positions 4, 6, 8, 12, 14,and 20 of its sense strand and in positions 21 and 22 of its antisensestrand, and phosphorothioate linkages between positions 21 and 22, and22 and 23, of the antisense strand and between position 20 and 21 of thesense strand; and

AL-DUP 5181 bears 2′-O-Me modifications in positions 4, 6, 8, 12, 14,and 20 of its sense strand and in positions 1, 6, 7, 13, 15, and 17 ofits antisense strand, and phosphorothioate linkages between positions 21and 22, and 22 and 23, of the antisense strand and between position 20and 21 of the sense strand.

The nucleotide sequences of siRNA duplexes AL-DUP 5048, AL-DUP 5167,AL-DUP 5168, AL-DUP 5169, AL-DUP 5170, AL-DUP 5182, and AL-DUP 5183 areidentical except that:

AL-DUP 5048 consists entirely of unmodified nucleotides;

AL-DUP 5167 bears 2′-O-Me groups in positions 21 and 22 andphosphorothioate linkages between positions 21 and 22, and 22 and 23, ofthe antisense strand, and a cholesterol moiety conjugated to the 3′-endof the sense strand;

AL-DUP 5168 bears 2′-O-Me groups in positions 3, 6, 8, 11, 15, and 18 ofits sense strand and positions 2, 3, 6, 8, 10, 13, 15, 18, and 21 of itsantisense strand, phosphorothioate linkages between positions 21 and 22,and 22 and 23, of the antisense strand, and a cholesterol moietyconjugated to the 3′-end of the sense strand;

AL-DUP 5169 bears 2′-O-Me groups in positions 2, 3, 6, 8, 10, 13, 15,18, and 21 of its antisense strand, phosphorothioate linkages betweenpositions 21 and 22, and 22 and 23, of the antisense strand, and acholesterol moiety conjugated to the 3′-end of the sense strand;

AL-DUP 5170 bears 2′-O-Me modifications in positions 3, 6, 8, 11, 15,and 18 of its sense strand and in positions 21 and 22 of its antisensestrand, phosphorothioate linkages between positions 21 and 22, and 22and 23, of the antisense strand, and a cholesterol moiety conjugated tothe 3′-end of the sense strand;

AL-DUP 5182 bears 2′-O-Me modifications in positions 3, 6, 8, 11, 15,and 18 of its sense strand and in positions 21 and 22 of its antisensestrand, and phosphorothioate linkages between positions 21 and 22, and22 and 23, of the antisense strand and between position 20 and 21 of thesense strand; and

AL-DUP 5183 bears 2′-O-Me modifications in positions 3, 6, 8, 11, 15,and 18 of its sense strand and in positions 2, 3, 6, 8, 10, 13, 15, 18,and 21 of its antisense strand, and phosphorothioate linkages betweenpositions 21 and 22, and 22 and 23, of the antisense strand and betweenposition 20 and 21 of the sense strand.

Stability of the siRNAs listed in Table 10 was tested in mouse and 95%human serum. Mouse serum was obtained from Sigma (Sigma-Aldrich ChemieGmbH, Taufkirchen, Germany, cat. No. M5905) or Charles River (CharlesRiver Laboratories, Sulzfeld, Germany, cat. No. MASER). Assay resultsreported herein were consistent among the different serum sourcestested. To test the stability of the modified siRNA in human serum,blood from eight human volunteers (270 mL) was collected and kept atroom temperature for 3 hours. The blood pool was then centrifuged at 20°C. and 3000 rcf using Megafuge 1.0 (Heraeus Instruments, KendroLaboratory Products GmbH, Langenselbold) to separate serum from thecellular fraction. The supernatant was stored in aliquots at −20° C. andused as needed. Human serum obtained from Sigma (Sigma-Aldrich ChemieGmbH, Taufkirchen, Germany, cat. No. H1513) was used in control assays.

Double stranded RNAs (300 pmol, ca. 4.2 μg) dissolved in 6 μl PBS wereadded to 60 μl human serum, and the mixture was incubated at 37° C. forvarying extents of time, e.g. 0, 15, or 30 minutes, or 1, 2, 4, 8, 16,or 24 hours. Subsequently, the whole tube containing the RNA/serumsolution was frozen in liquid nitrogen and stored at −80° C.

For analysis of non-cholesterol conjugated siRNAs, the frozen sampleswere placed on ice and 450 μl of 0.5 M NaCl was added. After completethawing, the solution was transferred to Phase-Lock Gel tubes(Eppendorf, Hamburg, Germany; cat. No. 0032 005.152), mixed with 500 μl50% phenol, 48% chloroform, 2% isoamylacohol (Carl Roth GmbH & Co KG,Karlsruhe, Germany, cat. No. A156.2), and an additional 300 μlChloroform were added. The tubes were vortexed vigorously for 30 secondsand subsequently centrifuged for 15 min at 16,200 rcf at 4° C. Theaqueous supernatant was transferred to a fresh tube and mixed with 40 μl3M Na-acetate pH 5.2, 1 μl GlycoBlue (Ambion, Tex., USA; cat. No. 9516)and 1 ml Ethanol 95%. RNA was precipitated overnight at −20° C.

Cholesterol-conjugated siRNAs were isolated by hot phenol-extraction inpresence of SDS (Sodium Dodecylsulfate). The serum sample (66 μl) wasmixed with 200 μl RNA buffer (0.5% SDS, 10 mM EDTA, 10 mM Tris pH7.5)and 200 μl water-saturated phenol (Carl Roth GmbH & Co KG Karlsruhe,Germany; cat. No. A980.1). The reaction tube was incubated for 20 min at65° C. In order to achieve phase separation, the tubes were placed onice for 5 min and subsequently centrifuged for 10 min at 16,200 rcf at4° C. The aqueous phase was transferred to a fresh tube. The remainingphenol phase was extracted a second time with 150 μl RNA portion andvigorous vortexing for 10 sec. The tubes were placed on ice for 2 minand then centrifuged for 10 min at 16,200 rcf at 4° C. The aqueous phaseof the second extraction was transferred and combined with thesupernatant of the first extraction. The RNA was precipitated by adding2 μl GlycoBlue (Ambion, Austin, Tex., USA; Cat. No. 9516) and 1 mlEthanol 95%. Precipitation of RNA was brought to completion overnight at−20° C.

Isolated RNA was analyzed by denaturing gel electrophoresis. Tubescontaining the precipitated RNA were centrifuged for 10 min at 16,200rcf at 4° C. The supernatant was removed and discarded. The RNA pelletwas washed with 400 μl 70% Ethanol, and re-pelleted by centrifugationfor 5 min at 16,200 rcf at 4° C. All liquid was removed and the pelletwas dissolved in 20 μl STOP buffer (95% formamide, 5% EDTA 0.5M, 0.02%xylene cyanol). The samples were boiled for 3 min at 92° C. and chilledquickly on ice. 10 μl were loaded on a denaturing 14% polyacrylamide gel(6M Urea, 20% formamide, Carl Roth GmbH & Co KG Karlsruhe, Germany). TheRNA was separated for about 2 h at 45 mA. RNA bands were visualized bystaining with the “stains-all” reagent (Sigma-Aldrich Chemie GmbH,Steinheim, Germany, cat. no. E9379) according to manufacturer'sinstructions.

While the unmodified AL-DUP 5024 was almost completely degraded after 1h of incubation with mouse and human serum, the modified siRNAs weremore resistant to degradation (see FIG. 3). Following electrophoreticseparation, full length RNA was stained with stains-all reagent for upto 3 hours for AL-DUP 5163, and up to 6 hours of incubation for AL-DUP5164, AL-DUP 5165, AL-DUP 5166, AL-DUP 5180, and AL-DUP 5181. Thegreatest stabilizing effect was seen in AL-DUP 5164, AL-DUP 5166, andAL-DUP 5181, indicating that the modification of sites prone todegradation in the sense strand was most effective. Additionalmodification of the antisense strand imparted only a small additionalstabilizing effect. (See FIG. 3)

Similarly, the unmodified AL-DUP 5048 was almost completely degradedafter 1 h of incubation with mouse serum, while the modified dsRNAs wereless sensitive to degradation. Following electrophoretic separation,full length RNA was stained with the stains-all reagent after up to 3hours for AL-DUP 5167, AL-DUP 5170, and AL-DUP 5182, and up to 6 hoursfor AL-DUP 5169, and up to 24 hours for AL-DUP 5168 and AL-DUP 5183.(See FIG. 3)

The siRNA duplexes listed in Table 10 were tested for their efficacy inreducing ApoB protein secretion into supernatant by cultured HepG2 cellsin order to select the most active duplexes for further examination invivo.

The silencing activity of cholesterol modified siRNAs specific for ApoBin in vitro assays in HepG2 cells was comparable to that of unmodifiedApoB-specific siRNAs. At 200 nM concentrations the two unconjugatedsiRNAs AL-DUP 5024 and AL-DUP 5048 reduced murine ApoB mRNA levels by84±9% and 72±9%, respectively, whereas the corresponding conjugated andmodified siRNAs AL-DUP 5167 and AL-DUP 5163 had an inhibitory activityof 61±8% % and 68±9%, respectively.

FIGS. 5A through 5L show dose-response curves of ApoB protein secretioninto supernatant of cultured human HepG2 cells incubated with mediacontaining 100, 33, 11, 3.7, 1.2, 0.4, 0.14, or 0.05 nM of theApoB-specific siRNA duplexes. The response is expressed as the ratio ofApoB protein concentrations in the supernatant of cells treated with theApoB-specific siRNA duplex to the ApoB concentration in the supernatantof cells treated with an unspecific control siRNA duplex. On the basisof these results, AL-DUP 5163, AL-DUP 5165, AL-DUP 5166 and AL-DUP 5167were chosen for testing in mice (see results below).

Example 7 Modified siRNA Duplexes Reduced ApoB mRNA Amounts in TissueSections from Liver and Jejunum, and ApoB Protein's Cholesterol Levelsin Serum of Male C57Bl/6 Mice

Bolus dosing of siRNAs in mice was performed by tail vein injectionusing a 27 G needle. SiRNAs were dissolved in PBS at a concentrationallowing the delivery of the intended dose in 8 μl/g body weight. Micewere kept under an infrared lamp for approximately 3 min prior to dosingto ease injection.

Pre-treatment blood samples were collected several days before dosing bycollecting 4-7 drops from the tail vein. Upon sacrifice byCO₂-asphyxiation, ca. 0.7 ml blood was collected by heart puncture, theliver and jejunum were collected, and tissue aliquots of 20-40 mg werefrozen in liquid nitrogen and stored at −80° C. until analysis.

ApoB100 mRNA levels were measured by branched-DNA-assay as describedabove. Triplicate samples of frozen tissue sections (liver or jejunum)of about 10-30 mg each were homogenized by sonication (Bandelin SonopulsHD 2070, BANDELIN electronic GmbH & Co. KG, Berlin, Germany) in 1 ml ofTissue and Cell Lysis solution (Epicentre, Madison, Wis., USA, cat. No.MTC096H) containing 84 μg/ml Proteinase K (Epicentre, Madison, Wis.,USA, cat. No. MPRK092) using 3-9 pulses of 0.9 sec each at an amplitudeof ca. 150 μm. Lysates were kept at −80° C. for at least 12 h(overnight) before analysis.

Frozen lysates were thawed at room temperature, and ApoB and GAPDH mRNAquantified using the Quantigene Explore Kit according to manufacturer'sinstructions. Nucleic acid sequences for Capture Extender (CE), LabelExtender (LE) and blocking (BL) probes were selected from the nucleicacid sequences of ApoB and GAPDH with the help of the QuantiGeneProbeDesigner Software 2.0 (Genospectra, Fremont, Calif., USA, cat. No.QG-002-02). Probe nucleotide sequences used in ApoB quantization areshown in Table 4. Probe nucleotide sequences used in GAPDH quantizationare shown in Table 6.

The ratio of ApoB mRNA to GAPDH mRNA in tissue samples was averaged overeach treatment group and compared to an untreated control group or acontrol group treated with an unrelated siRNA duplex.

ELISA assays were performed to quantitate the amount of ApoB100 proteinin mouse serum. To perform the assay, a 96 well plate was coated with100 μl of the mouse ApoB-100-specific monoclonal antibody LF3 (25 μg/ml;Zlot, C. H. et al., J. Lipid Res. 1999, 40:76-84) and the plate wasincubated for 2 hours at 37° C. The plate was washed three times withphosphate buffered saline (PBS) (PS Dulbecco without Ca²⁺, Mg²⁺,Biochrom AG, Berlin, Germany, cat. No. L182-05), and then the remainingbinding sites were blocked by adding 300 μl PBS containing 3% bovineserum albumin (BSA) (Carl Roth GmbH & Co KG, Karlsruhe, Germany, cat.no. 8076.2) to each well. Plates were incubated for 1 hour at roomtemperature. The plate was then washed 5 times with PBS. 0.2 μl mouseserum diluted in 100 μl PBS containing 0.1% Tween (Carl Roth GmbH & CoKG, Karlsruhe, Germany, cat. No. 9127.1) and 3% BSA was added to eachwell. After an incubation of 2 hours at 37° C. the plate was washed 5times with PBS. 100 μl of a 1:500 dilution of the polyclonal rabbitanti-mouse apolipoprotein B48/100 antibody (Acris Antibodies GmbH,Hiddenheim, Germany, cat. no. BP2050) was added to the wells andincubated for 2 hours at 37° C. After washing the plate 5 times withPBS, 100 μl of a donkey anti-rabbit IgG conjugated to horse radishperoxidase (Santa Cruz Biotechnology, Santa Cruz, Calif., USA, cat. no.sc2004) was added and incubated for 1 hour at 37° C. The plate waswashed 5 times with PBS and wells were incubated with 0.9 mg/ml OPD(o-phenylendiamine dihydrochloride, Merck Biosciences GmbH, Bad Soden,Germany cat. No. 523121) in 24 mmol/L citric acid buffer (Sigma-Aldrich,Taufkirchen, Germany, cat. no. C1909-1KG), pH 5.0 containing 0.03%hydrogen peroxide (Merck Biosciences GmbH, Bad Soden, Germany, cat. No.386790). The enzyme reaction was stopped by adding 0.5 mol/L H₂SO₄(Merck KgaA, Darmstadt, Germany, cat. No. 100731) and absorbance at 490nm was measured on a spectrophotometer (Perkin Elmer Wallac Victor3 1420multilabel reader, PerkinElmer LAS GmbH, Rodgau, Germany).

Total serum cholesterol in mouse serum was measured using theCholesterol FS reagent kit (DiaSys Diagnostic Systems GmbH, Holzheim,Germany) according to manufacturer's instructions. Measurements weretaken on a spectral photometer (DU 640B, Beckman Coulter GmbH,Unterschleiβheim, Germany).

S1-nuclease protection assay were used to detect siRNAs in liver andjejunum tissue and in serum following injections. Small pieces (10-30mg) of animal tissue were homogenized as described above for thebranched-DNA assay. These lysates were either processed immediately, orstored at −80° C. and thawed at room temperature prior to assayperformance. 100 μl lysate was transferred to a fresh reaction tube andmixed with 200 μl STE (Sodium chloride−TRIS−EDTA buffer; 500 mM NaCl, 9mM Tris pH 7.5, 0.9 mM EDTA) and 200 μl phenol (TRIS-EDTA saturatedphenol, Roti-phenol, Carl Roth GmbH & Co KG, Karlsruhe, Germany, cat.no. 0038.1). The tubes were vigorously mixed on a Vortex Genie 2(Scientific Industries, Inc., Bohemia, N.Y., USA, cat. no. SI-0256) atmaximum speed for 30 seconds, and subsequently centrifuged for 10 min at16,200 rcf and 4° C. About 310 μl aqueous supernatant was carefullyaspired and transferred to a new reaction tube, mixed with 50 μg E. colitRNA (Roche Diagnostics, Penzberg, Germany; cat. No. 109 541) and 900 μlEthanol 95%. Precipitation of RNA was continued over night at −20° C.

DNA probes for use in the S1-nuclease protection assays wereradioactively labelled. Probes of 25 to 27 nucleotides lengthcorresponded to the 21 nucleotide sense-strand sequence of the siRNAmolecules, but contained an additional 4 to 6 nucleotides at their3′-end serving as non-complementary extension. The DNA oligonucleotidesprobes were phosphorylated with γ-³²P ATP to introduce a radioactivephosphate group at their 5′-end. Fifteen picomoles of the respectiveprobe were mixed with 50 μCi of γ-³²P ATP (Amersham GE-Healthcare,Freiburg, Germany, cat. no. AA0018) and 10 U Polynucleotide kinase (NewEngland Biolabs, Frankfurt, Germany, MO201 S) were mixed in a totalvolume of 50 μl Polynucleotide kinase buffer (New England Biolabs,Frankfurt, Germany, cat. no. MO201S). This solution was incubated at 37°C. for 1 hour. The labelling reaction was terminated by passing thereaction mixture through a Microspin G-25 desalting column followinginstructions by the manufacturer (Amersham GE-Healthcare, Freiburg,Germany, cat. no. 27-5325-01). The resulting probe solutions were usedwithin 1-3 days.

To detect siRNAs from mouse tissue lysates, precipitated total RNA fromthe lysates was centrifuged for 10 min at 16,200 rcf and 4° C. Thesupernatant was carefully removed and discarded while keeping thenucleic acid pellet. This pellet was first resuspended in 50 μlS1-hybridization buffer (300 mM NaCl, 1 mM EDTA, 38 mM HEPES pH 7.0,0.1% Triton X-100) and then 1 μl of radioactive DNA probe solution wasadded. The hybridization reaction mixture was heated to 92° C. for 2min. The reaction tubes were immediately transferred to a heating blockkept at 37° C. and further incubated for 30 min. The hybridization wascontinued at room temperature for an additional 2 hours.

For the determination of siRNA concentrations in serum, 1 μl of serumwas mixed with 50 μl S1-hybridization buffer and 1 μl of radioactive DNAprobe, and the hybridization continued as above.

The following probes were used:

For AL-DUP 3001 and AL-DUP 5386:

SEQ. ID NO. 265 5′-

AACTGTGTGTGAGAGGTCCTTCTT-3′

For AL-DUP 5311

SEQ. ID NO. 266 5′-

TGATCAGACTCAATACGAATTCTTCTT-3′

For siRNAs derived from AL-DUP 5048 (AL-DUP 5048, AL-DUP 5167, AL-DUP5168, AL-DUP 5169, AL-DUP 5170, AL-DUP 5182, AL-DUP 5183, AL-DUP 5385,AL-DUP 5546)

SEQ. ID NO. 267 5′-

TCATCACACTGAATACCAATTCTTCT-3′

For siRNAs derived from AL-DUP 5024 (AL-DUP 5024, AL-DUP 5163, AL-DUP5164, AL-DUP 5165, AL-DUP 5166, AL-DUP 5180, AL-DUP 5181)

SEQ. ID NO. 268 5′-

GGTGTATGGCTTCAACCCTGTCTTCT-3′

For siRNAs derived from AL-DUP 5002 (AL-DUP 5536, AL-DUP 5537)

SEQ. ID NO. 245 5′-

ATTGATTGACCTGTCCATTCTCTTCTT-3′

For siRNAs derived from AL-DUP 5035 (AL-DUP 5538, AL-DUP 5539)

SEQ. ID NO. 246 5′-

ACCAACTTCTTCCACGAGTCTCTTCTT-3′

For siRNAs derived from AL-DUP 5089 (AL-DUP 5540, AL-DUP 5541)

SEQ. ID NO. 247 5′-

AGTTTGTGACAAATATGGGCTCTTCTT-3′

For siRNAs derived from AL-DUP 5097 (AL-DUP 5542, AL-DUP 5543)

SEQ. ID NO. 248 5′-

TTTACAAGCCTTGGTTCAGTTCTTCTT-3′

For siRNAs derived from AL-DUP 5098 (AL-DUP 5544, AL-DUP 5545)

SEQ. ID NO. 250 5′-

GAATCTTATATTTGATCCAATCTTCTT-3′

In addition, two probes hybridizing with micro-RNAs endogenouslyexpressed in liver (miRNA122) and jejunum (miRNA143) were used as aloading control.

For miRNA 122:

SEQ. ID NO. 269 5′-

AACACCATTGTCACACTCCATCTTCTT-3′

For miRNA 143:

SEQ. ID NO. 270 5′-

AGCTACAGTGCTTCATCTCATCTTCTT-3′

After hybridization, 450 μl of S1-nuclease digestion mix was added toeach tube (450 μl S1-reaction mix: 333 mM NaCl, 2.2 mM Zn-acetate, 66.7mM Na-acetate pH 4.5, 0.02% Triton X-100 and 100 U S1-Nuclease; AmershamGE-Healthcare, Freiburg, Germany; cat. no. E2410Y) to degrade anyunhybridized probe. The digestion reaction mixture was incubated at 37°C. for 30 min. The reaction digestion was terminated by the addition of30 μg tRNA (Roche Diagnostics, Penzberg, Germany; 109 541) in 7 μl of500 mM EDTA, pH 8.0, and 900 μl Ethanol 95%. The protected probes wereprecipitated at −20° C. over night or at −80° C. for 90 min.

Following precipitation, the protected probes were analyzed bydenaturing gel electrophoresis. The precipitated duplexed RNA wascentrifuged for 10 min at 16,200 rcf and 4° C. The supernatant wascarefully removed and discarded. The pellet was dissolved in 12 μl STOPbuffer (95% formamide, 5% EDTA 0.5M, 0.02% xylene cyanol). The tubeswere heated to 92° C. for 2 min and then immediately chilled on ice. 4μl of the solution were loaded per lane of a denaturing sequencing gel(12.5% acrylamide, 1× standard TBE buffer, 19 cm×20 cm×0.4 mmLength×Width×Depth; Rotiphorese DNA sequencing system, Carl Roth GmbH &Co KG, Karlsruhe, Germany, cat. no. A431.1). The gel was run for 45 minat 600 V corresponding to a voltage gradient of approximately 30 V/cm(EPS 3501XL, Amersham Biosciences, Uppsala, Sweden; cat. no.18-1130-05). The gel was dried on paper and exposed overnight to ageneral purpose phosphor screen imager (Amersham GE-Healthcare,Freiburg, Germany; cat. no. 63-0034-88). On the following day,radioactive bands were visualized on a Typhoon 9200 high performanceimager (Amersham GE-Healthcare, Freiburg, Germany; cat. no. 63-0038-49).Quantitation of radioactive band intensity was performed using theImageQuant TL software version 2003.01 supplied with Typhoon 9200 imagerby comparison to a dilution series of 60, 20, 6.6, and 2.2 fmol of therespective radioactive probe loaded onto the gel.

All animal experiments, except those involving animals transgenic forthe expression of human ApoB described below, were carried out incompliance with the regulations of the European Convention for theProtection of Vertebrate Animals used for Experimental and otherScientific Purposes. Male C57Bl/6 mice were obtained from Charles RiverLaboratories, Sulzfeld, Germany, and acclimatized for at least 5 daysbefore use. Animals were housed at 22±2° C. and 55±10% rel. humidity.Day/night rhythm was 12 hours, changing at 6:00 am (light) and 6:00 pm(dark). Animals were fed Ssniff R/M-H chow (Ssniff Spezialdiäten GmbH,Soest, Germany, cat. No. V1531) ad libitum, unless specificallyspecified otherwise below.

The following experimental protocols were performed.

A.) Three groups of 7 animals, age 3.5 months, received daily doses of50 mg/kg on three consecutive days of either AL-DUP 5163, AL-DUP 5166(sequences see Table 10), or an equivalent amount of carrier, and weresacrificed on the fourth day. Total serum cholesterol, serum ApoB100concentration, and liver and jejunum ApoB mRNA levels were determined.

The 2′-O-methyl modification of the nucleotides in positions 4, 6, 8,12, 14, and 20 in the sense strand of AL-DUP 5166 as compared to theotherwise identical AL-DUP 5163 afforded greater stability to AL-DUP5166 with respect to its degradation in serum (see FIG. 3). Thisexperiment was designed to test the ability of siRNA specific for mouseApoB to down-regulate the expression of the ApoB gene in the liver andjejunum of mice, and to lower ApoB protein levels and cholesterol levelsin serum. This experiment also tested whether an siRNA bearing 2′-O-Memodifications on its sense strand, which increases its stability inbiological serum was more potent in down-regulating the expression ofApoB, than an siRNA lacking the 2′OMe sense strand modification.

ApoB mRNA levels in liver and jejunum tissue were assayed by thebranched DNA assay. AL-DUP 5163 was found to lower the levels of ApoBmRNA in samples of liver tissue to 50±13% of the levels present in livertissue of control animals. Levels of ApoB mRNA in jejunum were loweredto 40±6% of the levels in control animals.

AL-DUP 5166 was found to lower the levels of ApoB mRNA in liver tissueto 59±9% of the mRNA levels in tissue of control animals. Levels of ApoBmRNA in jejunum were lowered to 14±3% of the levels in control animals.

B.) Two groups of 7 animals, age 3.5 months were treated for threeconsecutive days with daily doses of 50 mg/kg of either AL-DUP 5167(sequences see Table 10) or an equivalent amount of carrier. The micewere sacrificed on the fourth day. Total serum cholesterol, serum ApoB100 concentration, and liver and jejunum ApoB mRNA levels weredetermined.

ApoB mRNA levels were determined by branched DNA assay. AL-DUP 5167 wasfound to lower the levels of ApoB mRNA present in samples of livertissue from treated mice to 41±6% of the mRNA levels present in livertissue of control animals. Levels of ApoB mRNA in jejunum were loweredto 29±9% of the levels in control animals. Serum ApoB proteinconcentration in mouse sera was essentially unchanged at 101±9% ofcontrol levels. Serum cholesterol was lowered to 60±22% of carriercontrols.

C.) Three groups of 7 animals, age 2.5 months, received daily doses of50 mg/kg, on three consecutive days, of AL-DUP 5165 or an equivalentamount of carrier, and were sacrificed on the fourth day. Total serumcholesterol, serum ApoB 100 concentration, and liver ApoB mRNA levelswere determined.

The 2′-O-Me modification of the nucleotides in positions 1, 6, 7, 13,15, and 17 of its antisense strand of AL-DUP 5165 as compared to theotherwise identical AL-DUP 5163 (sequences see Table 10) affordedgreater stability to AL-DUP 5165 with respect to its degradation inserum (see FIG. 3), but the stabilizing effect was not quite as strongas seen in AL-DUP 5166. This experiment compared the ability of siRNAspecific for mouse ApoB, and bearing stabilizing modifications on theantisense strand, to down-regulate the expression of the ApoB gene inthe liver and jejunum of mice, and lower serum ApoB and cholesterollevels. The experiment also tested whether an siRNA modified to possessincreased stability in serum was more potent in down-regulating theexpression of ApoB.

The branched DNA assay was used to measure ApoB mRNA levels. AL-DUP 5165was found to lower the levels of ApoB mRNA in liver tissue from treatedmice to 68±12% of the mRNA levels present in liver tissue of controlanimals receiving carrier only. Serum ApoB protein concentration inmouse sera was lowered to 63±6% of control levels. Serum cholesterol wasfound unchanged at 99±26% of carrier control levels.

D.) Four groups of 6 animals, age 2.5 months, received daily doses of 50mg/kg, on three consecutive days, of either AL-DUP 5167, AL-DUP 3001,AL-DUP 5311, or an equivalent amount of carrier, and were sacrificed onthe fourth day. Total serum cholesterol, serum ApoB 100 concentration,and liver and jejunum ApoB mRNA levels were determined.

The nucleotide sequences of AL-DUP 5167 is shown in Table 10. Thesequences of AL-DUP 5311 and AL-DUP 3001 are as follows

AL-DUP 5311 SEQ. ID No. 271 Sense: 5′-

ugaucagacugaauacgaau(Chol)-3′ SEQ. ID No. 272 Antisense: 5′-

uucguauugagucugaucacmamc-3′ AL-DUP 3001 (SEQ. ID No. 273) Sense: 5′-

aacugugugugagagguccu(Chol)-3′ (SEQ. ID No. 274) Antisense: 5′-

ggaccucucacacacaguucgmcm-3′

AL-DUP 5311 represents a mouse ApoB mRNA mismatch siRNA to AL-DUP 5167where four G/C switches in positions 4, 10, 14, and 19 were made. ThissiRNA was a negative control for comparison with AL-DUP 5167.

AL-DUP 3001 represents an unrelated control siRNA. The sequence ofpositions 1 to 21 of the sense strand of AL-DUP 3001 corresponds tonucleotides 1252 to 1272 of cloning vector pGL3-Basic (Promega GmbH,Mannheim, Germany, cat. no. E1751), accession number U47295, and is partof a sequence encoding firefly (Photinus pyralis) luciferase. AL-DUP3001 was meant to serve as an additional negative control to AL-DUP5167.

This experiment was meant to confirm the earlier findings obtained withAL-DUP 5167, and to further show that the effects seen with AL-DUP 5167are sequence-specific.

ApoB mRNA levels were determined by branched-DNA assay. AL-DUP 5167 wasfound to lower the levels of ApoB mRNA in liver tissue from treated miceto 36±11% of the mRNA levels present in liver tissue of control animals.Levels of ApoB mRNA in jejunum were lowered to 27±8% of the levels incontrol animals. Serum ApoB protein concentration in mouse sera waslowered to approximately 29±16% of carrier control levels. Serumcholesterol levels were essentially unchanged at 73±35%.

AL-DUP 5311 was found to leave the levels of ApoB mRNA in liver tissuefrom treated mice essentially unchanged at 95±16% of the mRNA levelspresent in liver tissue of control animals injected with carrier. Levelsof ApoB mRNA in jejunum were found essentially unchanged at 120±19% ofthe levels in control animals. Serum ApoB protein concentration in mousesera was essentially unchanged at 109±76% of carrier control levels.Serum cholesterol levels were found essentially unchanged at 77±43% ofcarrier controls.

AL-DUP 3001 was found to leave the levels of ApoB mRNA in liver tissuefrom treated mice essentially unchanged at 79±22% of the mRNA levels inliver tissue of control animals receiving carrier only. Levels of ApoBmRNA in jejunum were found essentially unchanged at 130±33% of thelevels in control animals. Serum ApoB protein concentration in mousesera was found essentially unchanged at 104±55% of carrier controllevels. Serum cholesterol levels were found essentially unchanged at108±46% of carrier control levels.

E.) Seven groups of six animals, age 2.5 months, received daily doses of50 mg/kg, on three consecutive days, of either AL-DUP 5167, AL-DUP 3001,AL-DUP 5311, or an equivalent amount of carrier, or daily doses of 10mg/kg of AL-DUP 5167 on three consecutive days, or daily doses of 2mg/kg of AL-DUP 5167 on three consecutive days, or a single dose of 50mg/kg on day 1. The mice were sacrificed on the fourth day. Anothergroup of 6 animals received an osmotic pump implant (Alzet 1007D, ALZETOsmotic Pumps DURECT Corporation, Cupertino, Calif., USA) subcutaneouslyon their back slightly posterior to the scapulae on day 1. The pump wasset to deliver 0.5 μl/hr of a solution of 0.33 mg/μl AL-DUP 5167 for 7days, amounting to a daily dose of approximately 4 mg/kg body weight perday per animal. This group of animals was sacrificed on day 8. Totalserum cholesterol, serum ApoB 100 concentration, and liver and jejunumApoB mRNA levels were determined.

The nucleotide sequence of AL-DUP 5167, AL-DUP 5311 and AL-DUP 3001 aregiven above.

This experiment was meant to confirm the earlier findings obtained withAL-DUP 5167, using carrier, a mismatched siRNA (AL-DUP 5311), and anunrelated siRNA (AL-DUP 3001) as controls, and to further determinewhether bolus intravenous doses of 2 or 10 mg/kg body weight on threeconsecutive days, or a single dose of 50 mg/kg body weight on day 1,could suffice to elicit the effects seen when dosing 50 mg/kg bodyweight intravenously on three consecutive days in (C) and (D) above.Furthermore, this experiment set out to compare these dosing regimenswith continuous delivery of a lower dose of 4 mg/kg body weight per dayover 7 days from an osmotic pump.

At a dose of 50 mg/kg body weight, administered intravenously on threeconsecutive days followed by sacrifice on day 4, AL-DUP 5167 was foundto lower the levels of ApoB mRNA present in samples of liver tissue fromtreated mice to 69±17% of the mRNA levels present in liver tissue ofanimals receiving carrier only, and levels of ApoB mRNA in jejunum werelowered to 24±8% of the levels in control animals, as determined by thebranched-DNA assay. Serum ApoB protein concentration in mouse sera waslowered to 69±9% of carrier control levels. Serum cholesterol was foundessentially unchanged at 95±29% of carrier control levels.

At a dose of 10 mg/kg body weight, administered intravenously on threeconsecutive days followed by sacrifice on day 4, AL-DUP 5167 was foundto leave the levels of ApoB mRNA present in samples of liver tissue fromtreated mice essentially unchanged at 81±32% of the mRNA levels presentin liver tissue of animals receiving carrier only, and levels of ApoBmRNA in jejunum came to 62±13% of the levels in control animals, asdetermined by the branched-DNA assay. Serum ApoB protein concentrationin mouse sera was essentially unchanged at 101±19% of carrier controllevels. Serum cholesterol was found essentially unchanged at 101±29% ofcarrier control levels.

At a dose of 2 mg/kg body weight, administered intravenously on threeconsecutive days followed by sacrifice on day 4, AL-DUP 5167 was foundto leave the levels of ApoB mRNA present in samples of liver tissue fromtreated mice essentially unchanged at 109±38% of the mRNA levels presentin liver tissue of animals receiving carrier only, and levels of ApoBmRNA in jejunum came to 97±21% of the levels in control animals, asdetermined by the branched-DNA assay. Serum ApoB protein concentrationin mouse sera was essentially unchanged at 115±13% of carrier controllevels. Serum cholesterol was found essentially unchanged at 114±26% ofcarrier control levels.

At a dose of 50 mg/kg body weight, administered intravenously once onday 1 followed by sacrifice on day 4, AL-DUP 5167 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 41±20% of the mRNA levels present in liver tissue of animalsreceiving carrier only, and levels of ApoB mRNA in jejunum were loweredto 62±23% of the levels in control animals, as determined by thebranched-DNA assay. Serum ApoB protein concentration in mouse sera waslowered to 52±11% of carrier control levels. Serum cholesterol was foundessentially unchanged at 95±25% of carrier control levels.

At a dose of 50 mg/kg body weight, administered intravenously on threeconsecutive days followed by sacrifice on day 4, AL-DUP 5311 was foundto leave the levels of ApoB mRNA present in samples of liver tissue fromtreated mice essentially unchanged at 100±16% of the mRNA levels presentin liver tissue of animals receiving carrier only, and levels of ApoBmRNA in jejunum came to 100±20% of the levels in control animals, asdetermined by the branched-DNA assay. Serum ApoB protein concentrationin mouse sera was essentially unchanged at 97±11% of carrier controllevels. Serum cholesterol was found essentially unchanged at 129±37% ofcarrier control levels.

At a dose of 50 mg/kg body weight, administered intravenously on threeconsecutive days followed by sacrifice on day 4, AL-DUP 3001 was foundto leave the levels of ApoB mRNA present in samples of liver tissue fromtreated mice essentially unchanged at 97±28% of the mRNA levels presentin liver tissue of animals receiving carrier only, and levels of ApoBmRNA in jejunum came to 101±16% of the levels in control animals, asdetermined by the branched-DNA assay. Serum ApoB protein concentrationin mouse sera was lowered to 106±6% of carrier control levels. Serumcholesterol was found essentially unchanged at 129±43% of carriercontrol levels.

At a dose of 4 mg/kg body weight per day over 7 days delivered from anosmotic pump, followed by sacrifice on day 8, AL-DUP 5167 was found toleave the levels of ApoB mRNA present in samples of liver tissue fromtreated mice essentially unchanged at 79±24% of the mRNA levels presentin liver tissue of animals receiving carrier only, and levels of ApoBmRNA in jejunum came to 70±19% of the levels in control animals, asdetermined by the branched-DNA assay. Serum ApoB protein concentrationin mouse sera was lowered to 106±6% of carrier control levels. Serumcholesterol was found essentially unchanged at 129±43% of carriercontrol levels.

F.) Seven groups of six animals, age 2.5 months, received one bolus doseof 50 mg/kg on day 1 of AL-DUP 5167. A control group of six animalsreceived an equivalent amount of carrier. Groups of animals receivingsiRNA were sacrificed 12, 24, 36, 60, 84 and 108 hours post-dosing. Thecontrol group receiving carrier was sacrificed 84 hours post-dosing.Liver and jejunum ApoB mRNA levels were determined.

The nucleotide sequence of AL-DUP 5167 is given above.

This experiment was designed to yield the time course of the effects ofAL-DUP 5167 on ApoB mRNA levels in liver and jejunum, and on serum ApoBand cholesterol concentrations.

In animals sacrificed 12 hours post dosing of 50 mg/kg AL-DUP 5167intravenously, 100±32% of the ApoB mRNA levels present in liver tissueof animals receiving carrier only were found in the liver, and 145±78%of the ApoB mRNA levels present in jejunum tissue of animals receivingcarrier only were found in the jejunum. Serum ApoB protein concentrationin mouse sera was determined to 124±14% of carrier control levels.

In animals sacrificed 24 hours post dosing of 50 mg/kg AL-DUP 5167intravenously, 85±21% of the ApoB mRNA levels present in liver tissue ofanimals receiving carrier only were found in the liver, and 84±32% ofthe ApoB mRNA levels present in jejunum tissue of animals receivingcarrier only were found in the jejunum. Serum ApoB protein concentrationin mouse sera was determined to 92±52% of carrier control levels.

In animals sacrificed 36 hours post dosing of 50 mg/kg AL-DUP 5167intravenously, 64±20% of the ApoB mRNA levels present in liver tissue ofanimals receiving carrier only were found in the liver, and 88±19% ofthe ApoB mRNA levels present in jejunum tissue of animals receivingcarrier only were found in the jejunum. Serum ApoB protein concentrationin mouse sera was lowered to 55±12% of carrier control levels.

In animals sacrificed 60 hours post dosing of 50 mg/kg AL-DUP 5167intravenously, 73±10% of the ApoB mRNA levels present in liver tissue ofanimals receiving carrier only were found in the liver, and 41±13% ofthe ApoB mRNA levels present in jejunum tissue of animals receivingcarrier only were found in the jejunum. Serum ApoB protein concentrationin mouse sera was lowered to 43±16% of carrier control levels.

In animals sacrificed 84 hours post dosing of 50 mg/kg AL-DUP 5167intravenously, 72±13% of the ApoB mRNA levels present in liver tissue ofanimals receiving carrier only were found in the liver, and 68±22% ofthe ApoB mRNA levels present in jejunum tissue of animals receivingcarrier only were found in the jejunum. Serum ApoB protein concentrationin mouse sera was lowered to 54±15% of carrier control levels.

In animals sacrificed 108 hours post dosing of 50 mg/kg AL-DUP 5167intravenously, 68±15% of the ApoB mRNA levels present in liver tissue ofanimals receiving carrier only were found in the liver, and 85±15% ofthe ApoB mRNA levels present in jejunum tissue of animals receivingcarrier only were found in the jejunum. Serum ApoB protein concentrationin mouse sera was lowered to 51±8% of carrier control levels.

G.): Five groups of 10 animals, age 2.5 months, received daily doses of50 mg/kg body weight intravenously on three consecutive days of eitherAL-DUP 5167, AL-DUP 5385, AL-DUP 5311, AL-DUP 5386 or an equivalentamount of carrier, one group of 7 animals received AL-DUP 5163 by thesame dosing regimen, and all animals were sacrificed on the fourth day.Total serum cholesterol, serum ApoB 100 concentration, and liver andjejunum ApoB mRNA levels were determined. In addition, the amount ofsiRNA present in samples of liver and jejunum was approximated by the51-nuclease protection assay (see FIG. 6).

The nucleotide sequences of AL-DUP 5167, AL-DUP 5163, and AL-DUP 5311are given above. The nucleotide sequences of AL-DUP 5385 and AL-DUP 5386are:

AL-DUP 5385 SEQ. ID NO. 275 Sense: 5′-

ucaucacacugaauaccaau-3′ SEQ. ID NO. 276 Antisense: 5′-

uugguauucagugugaugacmamc-3′ AL-DUP 5386 SEQ. ID NO. 277 Sense: 5′-

aacugugugugagagguccu(Chol)-3′ SEQ. ID NO. 278 Antisense: 5′-

ggaccucucacacacaguucmgmc-3′

AL-DUP 5385 is identical to AL-DUP 5167, except that it bears nocholesterol moiety on the 3′-end of the sense strand, and has aphosphorothioate linkage between positions 20 and 21 of the sensestrand. The latter phosphorothioate group was meant to confer similarprotection towards exonucleolytic degradation as thephosphorothioate-bearing cholesterol modification (see FIG. 1).

AL-DUP 5386 is identical to AL-DUP 3001, except that the2′-O-methyl-modification in position 23 of the antisense strand wasremoved, and a 2′-O-methyl-modification was added in position 21. Thiswas believed to confer superior stabilization towards degradation ofAL-DUP 5386 over AL-DUP 3001.

This experiment was designed to confirm results obtained in (E) above,to further compare the activity of the cholesterol-conjugated AL-DUP5167 to the activity of the otherwise identical but cholesterol-lackingAL-DUP 5385, and to confirm that the lack of ApoB mRNA expressioninhibiting activity seen with AL-DUP 3001 was not due to rapiddegradation of AL-DUP 3001 in the serum of treated mice.

AL-DUP 5167 was found to lower the levels of ApoB mRNA present insamples of liver tissue from treated mice to 43±6% of the mRNA levelspresent in liver tissue of animals receiving carrier only, and levels ofApoB mRNA in jejunum were lowered to 27±10% of the levels in controlanimals, as determined by the branched-DNA assay. Serum ApoB proteinconcentration in mouse sera was lowered to 32±14% of carrier controllevels. Serum cholesterol concentration in mouse sera was lowered to63±11% of carrier control levels. Approx. 100-200 ng/of AL-DUP 5167 perg tissue was detected in liver and jejunum tissue samples by theS1-nuclease protection assay (FIG. 6A).

AL-DUP 5163 was found to lower the levels of ApoB mRNA present insamples of liver tissue from treated mice to 64±8% of the mRNA levelspresent in liver tissue of animals receiving carrier only, and levels ofApoB mRNA in jejunum were lowered to 49±13% of the levels in controlanimals, as determined by the branched-DNA assay. Serum ApoB proteinconcentration in mouse sera was lowered to 66±20% of carrier controllevels. Serum cholesterol concentration in mouse sera was essentiallyunchanged at 94±10% of carrier control levels. Approx. 50-150 ng/ofAL-DUP 5163 per g tissue was detected in liver and jejunum tissuesamples by the 51-nuclease protection assay.

AL-DUP 5385 was found to leave the levels of ApoB mRNA present insamples of liver tissue from treated mice essentially unchanged at84±12% of the mRNA levels present in liver tissue of animals receivingcarrier only, and levels of ApoB mRNA in jejunum came to 115±25% of thelevels in control animals, as determined by the branched-DNA assay.Serum ApoB protein concentration in mouse sera was found unchanged at101±24% of carrier control levels. Serum cholesterol concentration inmouse sera was essentially unchanged at 97±10% of carrier controllevels. AL-DUP 5385 remained undetectable in liver and jejunum tissuesamples in the S1-nuclease protection assay (FIG. 6A).

AL-DUP 5311 was found to leave the levels of ApoB mRNA present insamples of liver tissue from treated mice essentially unchanged at96±16% of the mRNA levels present in liver tissue of animals receivingcarrier only, and levels of ApoB mRNA in jejunum came to 96±28% of thelevels in control animals, as determined by the branched-DNA assay.Serum ApoB protein concentration in mouse sera was found unchanged at102±32% of carrier control levels. Serum cholesterol concentration inmouse sera was essentially unchanged at 104±10% of carrier controllevels. Approx. 50-200 ng/of AL-DUP 5311 per g tissue was detected inliver and jejunum tissue samples by the S1-nuclease protection assay(FIG. 6A).

AL-DUP 5386 was found to leave the levels of ApoB mRNA present insamples of liver tissue from treated mice essentially unchanged at89±11% of the mRNA levels present in liver tissue of animals receivingcarrier only, and levels of ApoB mRNA in jejunum came to 85±14% of thelevels in control animals, as determined by the branched-DNA assay.Serum ApoB protein concentration in mouse sera was found unchanged at94±31% of carrier control levels. Serum cholesterol concentration inmouse sera was essentially unchanged at 104±10% of carrier controllevels. Approx. 50-200 ng/of AL-DUP 5386 per g tissue was detected inliver and jejunum tissue samples by the S1-nuclease protection assay(FIG. 6A).

H.): Six groups of 6 animals, age 2.5 months, received a singleintravenous bolus dose of 100, 50, 25 or 12.5 mg/kg body weight ofAL-DUP 5167, or an equivalent amount of carrier. Animals were sacrificed72 h post-dosing. Total serum cholesterol, serum ApoB 100 concentration,and liver and jejunum ApoB mRNA levels were determined.

The nucleotide sequence of AL-DUP 5167 is given above in Table 10.

This experiment was undertaken to assess the dose response for AL-DUP5167.

At a dose of 100 mg/kg body weight, AL-DUP 5167 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 48±13% of the mRNA levels present in liver tissue of animalsreceiving carrier only, and levels of ApoB mRNA in jejunum were loweredto 37±3% of the levels in control animals, as determined by thebranched-DNA assay. Serum ApoB protein concentration in mouse sera waslowered to 57±14% of carrier control levels. Serum cholesterol waslowered to 71±14% of carrier control levels.

At a dose of 50 mg/kg body weight, AL-DUP 5167 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 79±15% of the mRNA levels present in liver tissue of animalsreceiving carrier only, and levels of ApoB mRNA in jejunum were loweredto 67±15% of the levels in control animals, as determined by thebranched-DNA assay. Serum ApoB protein concentration in mouse sera waslowered to 69±17% of carrier control levels. Serum cholesterol was foundessentially unchanged at 90±28% of carrier control levels.

At a dose of 25 mg/kg body weight, AL-DUP 5167 was found to leave thelevels of ApoB mRNA present in samples of liver tissue from treated miceessentially unchanged at 96±7% of the mRNA levels present in livertissue of animals receiving carrier only, and levels of ApoB mRNA injejunum were lowered to 56±11% of the levels in control animals, asdetermined by the branched-DNA assay. Serum ApoB protein concentrationin mouse sera were determined to 68±26% of carrier control levels. Serumcholesterol was found essentially unchanged at 93±8% of carrier controllevels.

At a dose of 12.5 mg/kg body weight, AL-DUP 5167 was found to leave thelevels of ApoB mRNA present in samples of liver tissue from treated miceessentially unchanged at 90±14% of the mRNA levels present in livertissue of animals receiving carrier only, and levels of ApoB mRNA injejunum were unchanged at 77±22% of the levels in control animals, asdetermined by the branched-DNA assay. Serum ApoB protein concentrationin mouse sera were determined to 95±5% of carrier control levels. Serumcholesterol was found essentially unchanged at 91±14% of carrier controllevels.

I): 8 groups of 6 animals, age 2.5 months, received a single intravenousbolus dose of 100 mg/kg body weight of AL-DUP 5167 (for sequence, seeTable 10), or an equivalent amount of carrier. Groups of animals treatedwith AL-DUP 5167 were sacrificed 18 h, 66 h, 96 h, 168 h, and 336 hpost-dosing; groups of animals treated with carrier were sacrificed 18h, 66 h, and 240 h post-dosing. The group sacrificed after 240 h wasused as the control, all values are expressed as percent of the averagefound in this group. Total serum cholesterol, serum ApoB 100concentration, and liver and jejunum ApoB mRNA levels were determined.An S1-nuclease protection assay was used to determine the amounts ofAL-DUP-5167 present in liver tissues.

This experiment was designed to confirm the time course of the effectsof AL-DUP 5167 on ApoB mRNA levels in liver and jejunum, and on serumApoB and cholesterol concentrations, and to extend the time ofobservation.

18 h post-dosing, 3.3 μg/g tissue of AL-DUP 5167 were recovered in liversamples, which dropped to 22 ng/g tissue after 66 h, and below the limitof detection thereafter.

In animals sacrificed 18 hours post dosing of 100 mg/kg AL-DUP 5167intravenously, 37±16% of the ApoB mRNA levels present in liver tissue ofthe 240 h carrier control group were found in the liver, and 87±29% ofthe ApoB mRNA levels present in jejunum tissue of the 240 h carriercontrol group were found in the jejunum. Serum ApoB proteinconcentration in mouse sera was lowered to XX±X % of carrier controllevels.

In animals sacrificed 66 hours post dosing of 100 mg/kg AL-DUP 5167intravenously, 47±7% of the ApoB mRNA levels present in liver tissue ofthe 240 h carrier control group were found in the liver, and 43±8% ofthe ApoB mRNA levels present in jejunum tissue of the 240 h carriercontrol group were found in the jejunum. Serum ApoB proteinconcentration in mouse sera was lowered to XX±X % of carrier controllevels.

In animals sacrificed 96 hours post dosing of 100 mg/kg AL-DUP 5167intravenously, 38±9% of the ApoB mRNA levels present in liver tissue ofthe 240 h carrier control group were found in the liver, and 78±14% ofthe ApoB mRNA levels present in jejunum tissue of the 240 h carriercontrol group were found in the jejunum. Serum ApoB proteinconcentration in mouse sera was lowered to XX±X % of carrier controllevels.

In animals sacrificed 168 hours post dosing of 100 mg/kg AL-DUP 5167intravenously, 57±5% of the ApoB mRNA levels present in liver tissue ofthe 240 h carrier control group were found in the liver, and 87±27% ofthe ApoB mRNA levels present in jejunum tissue of the 240 h carriercontrol group were found in the jejunum. Serum ApoB proteinconcentration in mouse sera was lowered to XX±X % of carrier controllevels.

In animals sacrificed 336 hours post dosing of 100 mg/kg AL-DUP 5167intravenously, 94±10% of the ApoB mRNA levels present in liver tissue ofthe 240 h carrier control group were found in the liver, and 109±12% ofthe ApoB mRNA levels present in jejunum tissue of the 240 h carriercontrol group were found in the jejunum. Serum ApoB proteinconcentration in mouse sera was lowered to XX±X % of carrier controllevels.

In animals sacrificed 18 hours post dosing of saline control AL-DUP 5167intravenously, 83±21% of the ApoB mRNA levels present in liver tissue ofthe 240 h carrier control group were found in the liver, and 109±26% ofthe ApoB mRNA levels present in jejunum tissue of the 240 h carriercontrol group were found in the jejunum. Serum ApoB proteinconcentration in mouse sera was lowered to 51±8% of carrier controllevels.

In animals sacrificed 18 hours post dosing of 100 mg/kg AL-DUP 5167intravenously, 104±19% of the ApoB mRNA levels present in liver tissueof the 240 h carrier control group were found in the liver, and 97±21%of the ApoB mRNA levels present in jejunum tissue of the 240 h carriercontrol group were found in the jejunum. Serum ApoB proteinconcentration in mouse sera was lowered to 51±8% of carrier controllevels.

This experiment shows that the action of a cholesterol-conjugated siRNAmay persist for 7 days or more in the liver, and 3 days or more in thegut. The latter is consistent with the average lifespan of theintestinal enterocyte.

Conclusions from experiments A)-I): An important consideration for siRNAbased inhibition to gene expression is whether the observed effects arespecific and not due to “off target” effects and potential interferonresponses that have been reported with siRNAs in vitro and otheroligonucleotide-based approaches. In our experiments, the effects ofApoB-specific, cholesterol-conjugated siRNAs were seen with severalindependent siRNAs targeting separate sequence regions of the ApoB mRNA.Further, the in vivo silencing of ApoB was specific as neither anunspecific siRNA nor a mismatch control siRNA mediated a significantreduction in ApoB mRNA, plasma ApoB protein levels, or totalcholesterol. Cholesterol-conjugated ApoB-specific siRNAs, but notunconjugated ApoB-specific siRNAs, showed biological activity,demonstrating an important role for cholesterol conjugation to achievesystemic in vivo activity and suggesting the opportunity to furtheroptimize activity based on systemic administration through chemicalconjugation strategies.

Example 8 Cholesterol Stabilizes siRNA Activity

In exploring the potential for synthetic siRNAs to silence endogenoustarget genes in vivo, we have found that chemically-stabilized andcholesterol-conjugated siRNAs have markedly improved pharmacologicproperties in vitro and in vivo. Chemically stabilized siRNAs withpartial phosphorothioate backbone and 2′-O-methyl modifications on thesense and antisense strands showed significantly enhanced resistancetowards degradation by exo- and endonucleases in serum and tissuehomogenates. The conjugation of cholesterol at the 3′-end of the sensestrand of a siRNA via a pyrrolidine linker (thereby generatingcholesterol-conjugated siRNA) did not result in a significant loss ofgene silencing activity in cell culture. In HeLa cells transientlyexpressing luciferase from a transfected plasmid and in the absence oftransfection reagent or electroporation only a cholesterol-conjugatedsiRNA inhibited luciferase expression (IC₅₀ 200 nM) whereas unconjugatedsiRNA was inactive. Binding of cholesterol-conjugated siRNAs to humanserum albumin (HSA) was determined by surface plasmon resonancemeasurement; unconjugated siRNAs demonstrated no measurable binding toHSA, while cholesterol-conjugated siRNAs were found to bind to HSA withan estimated K_(D)=1 μM. Due to enhanced binding to serum proteins,cholesterol-conjugated siRNAs administered to rats by IV injectionshowed improved in vivo pharmacokinetic properties as compared tounconjugated siRNAs. Following IV injection in rats at 50 mg/kg,radioactively-labeled cholesterol-conjugated siRNAs had at_(1/2)=95+/−13 min in plasma whereas unconjugated siRNAs had a plasmat_(1/2)=6.2+/−0.6 min, as determined by curve fitting simulationassuming a two compartment model, first order elimination rate, usingWinNonLin 4.1 (Pharsight Corporation, Mountain View, Calif., USA). Asmeasured by RNase protection assay, cholesterol-conjugated siRNAs showedbroad tissue biodistribution 24 h after a single 50 mg/kg IV bolusinjection in mice. Whereas no detectable amounts of unconjugated siRNAswere observed in tissue samples, significant levels ofcholesterol-conjugated siRNAs of about 200 ng/g tissue were detected inliver, heart, kidney, and lung samples. Together, these studiesdemonstrated that cholesterol conjugation significantly improves in vivopharmacokinetic properties of siRNAs.

Example 9 ApoB Expression in Human ApoB-100 Transgenic Mice is Reducedby siRNAs Specific for Human and Mouse ApoB

The experimental procedures were approved by the Alnylam InstitutionalAnimal Care and Use Committee, and were performed in accordance withcity of Cambridge, Mass. regulations regarding animal welfare.

Hemizygous male Human ApoB-100 transgenic mice (strain designation:B6.5SJL-Tg(APOB100)N20) were obtained from Taconic (Taconic, Germantown,N.Y., USA, cat. no. 1004-T) and were housed at constant temperature andhumidity on a 12 hr light/dark cycle (6:30 AM/6:30 PM). Animals were fedirradiated standard rodent chow (PicoLab® Rodent Diet 20, Purina Mills,LLC, St. Louis, Mo., USA, cat. no. 5053).

Animals at 30-32 weeks of age were divided into three groups of eightfor treatment. One group received three daily tail vein injections (24hours between doses) of phosphate buffered saline (10 μl per gram bodyweight). A second group received three daily tail vein injections (24hours between injections) of 50 mg siRNA AL-DUP 5167 per kilogram bodyweight in a dosing volume of 10 μl per gram body weight. The third groupreceived three daily tail vein injections (24 hours between injections)of 50 mg siRNA AL-DUP 5311 per kilogram body weight in a dosing volumeof 10 μl per gram body weight. The siRNA duplexes were formulated inphosphate buffered saline.

Twenty-four hours after the final injection, animals were sacrificed byCO₂ asphyxiation. Whole liver as well as the segment of the smallintestine corresponding to the jejunum was harvested from each animaland rapidly frozen in liquid nitrogen. Frozen tissues were ground to afine powder using a mortar and pestle.

Approximately 10 mg of each tissue powder was added to an ice-cold 1.5ml Eppendorf tube, and 1 ml Tissue and Cell Lysis Solution (Epicentre,Madison, Wis., USA, cat. No. MTC096H) containing 3.3 μl (10 μl per 3 ml)of a 50 μg/μl stock solution of Proteinase K (Epicentre, Madison, Wis.,USA, cat. No. MPRK092) were added. The tubes were vortexed and incubatedat 65° C. for 15 minutes; vortexing every 5 minutes. Cellular debris waspelleted at 5000 rcf for 10 minutes at RT, and 800 μl supernatant weretransferred to a fresh tube. Lysates were used immediately in thebranched DNA assay (described above) to determine relative levels ofApoB and GAPDH mRNA, or stored at −80° C. for later use.

The ApoB specific siRNA AL-DUP 5167 was found to reduce mouse ApoB mRNAlevels (significantly different at p<0.01); 43±10% in liver and 58±12%in jejunum of mouse ApoB mRNA levels in carrier treated animals werefound in animals treated with AL-DUP 5167. Human ApoB mRNA in liver wasreduced to 40±10% of levels in livers of control animals. The mismatchcontrol siRNA AL-DUP 5311 was found to leave ApoB mRNA levelsessentially unchanged; 93±20% in liver and 104±13% in jejunum of mouseApoB mRNA levels in carrier treated animals were found in animalstreated with AL-DUP 5167. Human ApoB mRNA in liver was determined to92±24% of levels in livers of control animals.

Example 10 Specific ApoB Cleavage Sites can be Identified by 5′-RACE PCR

Primers were purchased from Operon Biotechnologies, Inc. (Alameda,Calif., USA).

The specific siRNA-induced cleavage products of ApoB mRNA in pooledliver and jejunum from each of the treatment groups of experiment (G.)above (Example 7) were identified by 5′-RACE as described in Llave, etal. Science 2002, 297:2053-6, and Yekta, et al. Science 2004, 304:594-6,with the following modifications and primers given below. In suchexperiment, an adaptor is reacted with 5′-phosphate-bearing RNA presentin an RNA sample, such as the 3′-products of the cleavage of mRNA bysiRNA-complexed RISC. The products of most, if not all, nucleolyticreactions catalyzed by nucleases do not contain a 5′-phosphate group andtherefore will not react with the adaptor. In the subsequent PCRreactions, only those molecular species comprising both the adaptorsequences as well as the target gene sequence are amplified byappropriate selection of primers.

Following ligation of the RACE adapter (“GeneRacer” adapter,Invitrogen), cDNA synthesis was primed using a gene specific primer,5167GSP, to yield “5167” cDNA. Sequences corresponding to ApoB wereamplified in sequential PCR reactions using the following primer pairs:GR5′-XbaI(forward)+5167 ApoB Rev2-SalI(reverse)  PCR reaction 1GS5′Nest F-XbaI(forward)+5167 ApoB Rev3-SalI(reverse)  PCR reaction 2

A fifty-fold dilution of PCR reaction 1 was used in PCR reaction 2.Products of each PCR reaction were analyzed by agarose gelelectrophoresis, and visualized by ethidium bromide staining Specificbands of the expected size corresponding to siRNA-directed cleavage wereseen in liver from animals receiving AL-DUP 5167 and in jejunum fromanimals receiving AL-DUP 5167 and, to a lesser extent, AL-DUP 5385 (seeFIG. 7).

The specific bands from PCR reaction 1 were excised and sequenced(sequencing primer: 5167 ApoB Rev3-SalI) to confirm the presence of thejunction between the RACE adapter and nucleotide 10226 of mouse ApoB(Accession number: XM137955).

To specifically amplify fragments corresponding to the predicted siRNAcleavage site, PCR reaction 1 was diluted fifty fold and amplified withthe following primer pairs:iApoB 5167-XbaI(forward)+5167 ApoB Rev3-SalI(reverse)  PCR reaction 3

A PCR product is formed in PCR reaction 3 if and only if a reactionproduct is present in PCR reaction 1 combining the RACE adaptor with theRISC cleavage product of ApoB mRNA predicted by RNA interferencemediated by AL-DUP 5167. Products of this PCR reaction were visualizedas described above (FIG. 7). Confirmatory sequencing of the amplifiedbands was performed as above.

Primer Sequences:

GR5′-XbaI SEQ. ID NO. 279 5′-

TCTAGAGCGACTGGAGCACGAGGACACTGA-3′ GS5′Nest F-XbaI SEQ. ID NO. 280 5′-

TCTAGAGGGACACTGACATGGACTGAAGGAGTA-3′ 5167 GSP SEQ. ID NO. 281 5′-

TCCTGTTGCAGTAGAGTGCAGCT-3′ 5167 ApoB Rev2-Sal I SEQ.ID NO. 282 5′-

CGCGTCGACGTGGGAGCATGGAGGTTGGCAGTTGTTC-3′ 5167 ApoB Rev3-Sal ISEQ. ID NO. 283 5′-

CGCGTCGACGTAATGGTGCTGTCATGACTGCCCTT-3′ iApoB 5167-Xba I SEQ. ID NO. 2845′-

TCTAGAGCATGGACTGAAGGAGTAGAAAGAA-3′

Example 11 Further Testing of Modified siRNAs

Design of further modified siRNAs

Further siRNAs representing modified versions of AL-DUP 5002, AL-DUP5035, AL-DUP 5048, AL-DUP 5089, AL-DUP 5097, and AL-DUP 5098 were testedfor stability and activity towards inhibiting the expression of ApoB. Amodified version of AL-DUP 5048 was synthesized bearing a cholesterylmoiety linked to the 3′-end of the sense strand via a pyrrolidinelinker. For each of the unmodified iRNA agents AL-DUP 5002, AL-DUP 5035,AL-DUP 5089, AL-DUP 5097, and AL-DUP 5098, one iRNA agent wassynthesized with a 21-nucleotide sense strand, a 23-nucleotide antisensestrand forming a 2-nucleotide 3′-overhang, bearing a cholesteryl moietyon the 3′-end of the sense strand linked via aphosphorothioate-comprising linker, 2′-O-methyl nucleotides in positions21 and 22 of the antisense strand, and phosphorothioate linkages betweenpositions 21 and 22, and 22 and 23, of the antisense strand. Thisconfiguration corresponds to the pattern of modifications already usedin AL-DUP 5167, and is meant to protect the iRNA agent from thedegrading activity of exonucleases.

In addition, a second iRNA agent was synthesized representing a modifiedversion of AL-DUP 5002, AL-DUP 5035, AL-DUP 5089, AL-DUP 5097, andAL-DUP 5098, bearing a cholesteryl moiety on the 3′-end of the sensestrand linked via a phosphorothioate-comprising linker, 2′-O-methylmodified nucleotides in the positions of the 5′-most pyrimidines in alloccurrences of the sequence motifs 5′-UA-3′, 5′-CA-3′, 5′-UU-3′, and5′-UG-3′, and phosphorothioate linkages between positions 21 and 22, and22 and 23, of the antisense strand. These additional modifications weremade to protect these siRNAs from the degrading activity ofendonucleases. The corresponding sequences are listed in Table 11.

TABLE 11 Modified iRNA agents for stability and activity assays SEQ.SEQ. Duplex ID ID descriptor No. Sense strand sequence No.Antisense strand sequence AL-DUP 5546 285

ucaucacacugaauaccaau(chol) 286

aggguugaagccauacaccumcmu AL-DUP 5536 287

aumumgaumumgaccumguccmaumuc(chol) 288

aaumggacmaggucaaucmaaucmumu AL-DUP 5537 289

auugauugaccuguccauuc(chol) 290

aauggacaggucaaucaaucmumu AL-DUP 5538 291cmaccmaacumucumuccmacgaguc(chol) 292 gacucgumggaagaagumumggumgmumuAL-DUP 5539 293 caccaacuucuuccacgaguc(chol) 294gacucguggaagaaguuggugmumu AL-DUP 5540 295gagumumumgumgacmaaaumaumgggc(chol) 296 gcccmaumaumumumgucmacmaaacucmcmaAL-DUP 5541 297 gaguuugugacaaauaugggc(chol) 264gcccauauuugucacaaacucmcma AL-DUP 5542 262cumumumacmaagccumumggumucmagu(chol) 263 acumgaaccmaaggcumumgumaaagmumgAL-DUP 5543 260 cuuuacaagccuugguucagu(chol) 259acugaaccaaggcuuguaaagmumg AL-DUP 5544 258ggaaucumumaumaumumumgauccmaa(chol) 257 umumggaucmaaaumaumaagaumuccmcmuAL-DUP 5545 252 ggaaucuuauauuugauccaa(chol) 251uuggaucaaauauaagauuccmcmu m = 2′O-methyl modification; “(chol)”indicates cholesterol conjugated to the 3′-end via a pyrrolidine linkercomprising a phosphorothioate; “(chol)” indicates cholesterol conjugatedto the 3′-end via a pyrrolidine linker lacking a phosphorothioate

siRNA Stability Testing

The siRNAs, the sequences of which are shown in Table 11, were testedfor stability by incubation in human serum (Sigma-Aldrich Chemie GmbH,Taufkirchen, Germany, cat. No. H1513) followed by isolation ofseparation of fragments by HPLC. A 50 μM solution of the respectivesiRNA in phosphate buffered saline (PBS, Sigma-Aldrich Chemie GmbH,Taufkirchen, Germany) was incubated with serum at a ratio of 10:1serum:siRNA solution for 30 min, 1, 2, 4, 8, 16 and 24 hours, andsamples were analysed as described below.

Determination of siRNA degradation time course by HPLC followingProteinase K treatment of serum samples

Proteinase K (20 mg/ml) was obtained from peQLab (Erlangen, Germany;Cat.-No. 04-1075) and diluted 1:1 with deionized water (18.2 mΩ) to afinal concentration of 10 mg/ml Proteinase K. Proteinase K Buffer (4.0ml TRIS-HCl 1M pH 7.5, 1.0 ml EDTA 0.5M, 1.2 ml NaCl 5M, 4.0 ml SDS 10%)was prepared fresh and kept at 50° C. until use to avoid precipitation.

A 40 mer of poly(L-dT), (L-dT)₄₀ was obtained from Noxxon Pharma AG(Berlin, Germany) and used as an internal standard. Polymers of theL-enantiomers of nucleic acids show an extraordinary stability towardsnucleolytic degradation (Klussman S, et al., Nature Biotechn. 1996,14:1112) but otherwise very similar properties when compared tonaturally occurring nucleic acids consisting of R-enantiomers.

To terminate the siRNA-degradation, 25 μl of Proteinase K buffer wereadded to serum incubation samples immediately after expiry of therespective incubation period, the mixture vortexed at highest speed for5 s (Vortex Genie 2, Scientific Industries, Inc., Bohemia, N.Y., USA,cat. no. S10256), 8 μl Proteinase K (10 mg/ml) were added followed byvortexing for 5 s, and finally the mixture was incubated for 20 min in athermomixer at 42° C. and 1050 rpm.

5 μl of a 50 μM solution (250 pmole) of (L-dT)₄₀ were added as aninternal standard to each well, the solution was vortexed for 5 s, andthe tube centrifuged for 1 min in a tabletop centrifuge to collect alldroplets clinging to the inner surfaces of the wells at the bottom. Thesolution was transferred to a 96 well Captiva 0.2 um filter plate(Varian, Germany, Cat. No. A5960002) and filtered by centrifugation at21900 rcf for 45 min.

The incubation wells were washed with 47.5 μl deionized water (18.2 ma),the wash filtered through the Captiva Filter Unit at 21900 rcf for 15min, and the wash step repeated. Approximately 180 μl of the theoreticaltotal volume of 200 μl are on average recovered after the second washingstep.

Ion exchange chromatographic separation of siRNA single strands fromeach other and from degradation products:

A Dionex BioLC HPLC-system equipped with inline-degasser, autosampler,column oven and fixed wavelength UV-detector (Dionex GmbH, Idstein,Germany) was used under denaturing conditions. Standard run parameterswere:

-   Column: Dionex DNA-Pac100; 4×250 mm-   Temperature: 75° C.-   Eluent A: 10 mM NaClO₄, 20 mM TRIS-HCl, 1 mM EDTA; 10% acetonitrile,    pH=8.0-   Eluent B: 800 mM NaClO₄, 20 mM TRIS-HCl, 1 mM EDTA; 10%    acetonitrile, pH=8.0-   Detection: @260 nm-   Gradient: 0-1 min: 10% B    -   1-11 min: 10%→35% B    -   11-12 min: 35% B→100% B    -   12-14 min: 100% B→10% B    -   14-16 min: 10% B for column reequilibration-   Injection volume: 20 μl

Where separation between the two strands of an siRNA was notsatisfactory or a degradation fragment co-eluted with one strand, thechromatographic parameters were adjusted by changing temperature, pH,replacement of NaClO₄ by NaBr, the concentration of acetonitrile, and/oradjusting the slope of the eluent gradient until separation was achievedwhich allowed separate quantitation of the peaks from sense andantisense strand.

Peak areas for full length strands were obtained by integration of theUV detector signal using software supplied by the manufacturer of theinstrument (Chromeleon 6.6; Dionex GmbH, Idstein, Germany).

Data Analysis:

Integrated sense strand, antisense strand, and internal standard peakareas were obtained for all samples and the normalization control.

A correction factor CF, accounting for liquid losses in the filtrationand washing steps, was determined for every sample by calculating theratio of experimental to theoretical internal standard peak area. Thetheoretical internal standard peak area is obtained, e.g. from acalibration curve of the internal standard obtained by injecting 50 μleach of a serial dilution of the 50 μM solution of (L-dT)₄₀ onto theHPLC column, and calculation of the theoretical peak area correspondingto 25 pmole (L-dT)₄₀ with the equation obtained by linear least squarefit to the peak areas from the dilution series. The correction factor CFto be applied to the peak areas of the sense and antisense strand is theobtained as:CF=PeakArea_(intStd)(theoretical)/PeakArea_(intStd)(Sample)

This treatment assumes that, by virtue of washing the filter twice,virtually complete recovery is achieved in the combined filtrates, andcorrects for the variable volume of wash water retained in the filter,such that peak areas from different samples can be compared.

The peak areas obtained for the sense and antisense strand peaks foreach time point are then multiplied with the correction factor CF toobtain Normalized Peak Areas (NPA_(sense,t), NPA_(antisense,t)):NPA_(sense or antisense,t)=(Peak Area_(sense or antisense,t))×CF

To obtain the relative amount of remaining Full Length Product (% FLP)for the sense and antisense strands at time t, the Normalized Peak Areafor each strand at time t=0 min (NPA_(sense,t)=₀, NPA_(antisense,t=0))is set as 100%, and the NPAs from other time points are divided by thesevalues.% FLP_(t=1,2,3 . . . n)=(NPA_(t=1,2,3 . . . n)/NPA_(t=0))*100

The value obtained from the control sample, where the siRNA wasincubated with annealing buffer only, may serve as a control of theaccuracy of the method. The % FLP for both strands should lie near 100%,within error margins, regardless of time of incubation.

The degradation half life t_(1/2) may then be calculated for eachstrand, assuming first order kinetics, from the slope of a linear leastsquare fit to a plot of ln(% FLP) versus time as:t _(1/2)=ln(0,5)/slope

Serum half lifes of siRNAs described by the sequences in Table 11

The degradation half lifes of the full length products of the siRNAsdescribed by the sequences shown in Table 11 are given in Table 12. Asis evident from the difference in the half life of the antisense strandof AL-DUP 5546 compared to the half life of its sense strand or theantisense strand of AL-DUP 5167, protecting the 3′-end of a strand bymeans of 2′-O-methyl groups and phosphorothioate linkages in the3′-penultimate nucleotides affords an increase of approximately 6- to7-fold in terms of the degradation half life. Further substituting2′-O-methyl modified nucleotides at sites particularly prone toendonucleolytic degradation further improved half lifes by approximately3- to 4-fold, except for AL-DUP 5543, where the average-fold improvementwas 20.

TABLE 12 Serum half lifes of siRNAs with different stabilizingmodifications t_(1/2) t_(1/2) (sense strand) (antisense strand)average-fold Duplex descriptor [h] [h] improvement¹ AL-DUP 5167 8.7 6.54 AL-DUP 5546 6.8 0.9 AL-DUP 5536 22.7 16.6 3 AL-DUP 5537 7.4 7.7 AL-DUP5538 21.1 18.4 4 AL-DUP 5539 6.3 3.7 AL-DUP 5540 27.3 24.7 3 AL-DUP 55418.2 9.0 AL-DUP 5542 40.3 15.9 20 AL-DUP 5543 1.5 1.2 AL-DUP 5544 17.514.9 3 AL-DUP 5545 5.7 6.8 ¹[((t_(1/2)(modified sensestrand)/t_(1/2)(unmodified sense strand)) + (t_(1/2)(modified antisensestrand)/t_(1/2)(unmodified antisense strand)))/2]

In vitro activity of siRNAs modified to resist endonucleolyticdegradation

In vitro activity of the siRNAs of Table 11 was tested as described inExample 3 hereinabove. Results are shown in Table 13.

TABLE 13 In vitro activity of siRNAs modified to resist endonucleolyticdegradation compared to IC₅₀ modified IC₅₀ unmodified Duplex descriptor[nM] Duplex descriptor [nM] AL-DUP 5167 0.4 AL-DUP 5546 0.5 AL-DUP 55360.6 AL-DUP 5537 0.6 AL-DUP 5538 21 AL-DUP 5539 1 AL-DUP 5540 7 AL-DUP5541 7 AL-DUP 5542 3 AL-DUP 5543 6 AL-DUP 5544 7 AL-DUP 5545 4

As is evident from the comparison of the IC₅₀ for AL-DUP 5167 and AL-DUP5546 in Table 13, the introduction of phosphorothioate linkages betweenpositions 21 and 22, and 22 and 23, and 2′-O-methyl groups in positions21 and 22, of the antisense strand, in AL-DUP 5167 did not adverselyaffect the activity of this siRNA. Furthermore, as can be seen from acomparison of the IC₅₀ for AL DUP 5536 vs. AL-DUP 5537, AL DUP 5538 vs.AL DUP 5539. AL DUP 5540 vs. AL-DUP 5541, AL DUP 5542 vs. AL-DUP 5543,and AL DUP 5544 vs. AL DUP 5545, the introduction of 2′-O-methylmodified nucleotides in the positions of the 5′-most pyrimidines in alloccurrences of the sequence motifs 5′-UA-3′, 5′-CA-3′, 5′-UU-3′, and5′-UG-3′ in most cases had no adverse impact on the activity of thesemolecules either.

In vivo activity of siRNAs modified to resist endonucleolyticdegradation

The following experiment was performed using routines and procedures asdescribed in Example 7 above.

13 groups of 5 animals, age 2.5 months, received a single intravenousbolus dose of 100 mg/kg body weight of AL-DUP 5167, AL DUP 5536, AL-DUP5537, AL DUP 5538, AL DUP 5539, AL DUP 5540, AL-DUP 5541, AL DUP 5542,AL-DUP 5543, AL DUP 5544, AL DUP 5545, or an equivalent amount ofcarrier. Animals were sacrificed 72 h post-dosing. Total serumcholesterol, serum ApoB 100 concentration, and liver and jejunum ApoBmRNA levels were determined. In addition, the concentration of the siRNAwas determined in liver, jejunum, and serum samples from 3 animals fromeach group by the S1-nuclease protection assay as described in Example7; however, quantitation of radioactive band intensity was performed byvisual comparison of bands to the dilution series, and standarddeviations were not calculated.

The nucleotide sequence of AL-DUP 5167 is given above in Table 10. Thenucleotide sequences of AL DUP 5536, AL-DUP 5537, AL DUP 5538, AL DUP5539, AL DUP 5540, AL-DUP 5541, AL DUP 5542, AL-DUP 5543, AL DUP 5544,and AL DUP 5545 are given above in Table 11.

This experiment was undertaken to assess the impact of modificationsintroduced into siRNAs to improve their stability in biological media ontheir gene expression inhibiting activity in vivo.

At a dose of 100 mg/kg body weight, AL-DUP 5167 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 42±12% of the mRNA levels present in liver tissue of animalsreceiving carrier only, and levels of ApoB mRNA in jejunum were loweredto 45±8% of the levels in control animals, as determined by thebranched-DNA assay. Serum ApoB protein concentration in mouse sera waslowered to 44±23% of carrier control levels. Serum cholesterol was leftessentially unchanged at 75±20% of carrier control levels. Average iRNAconcentrations for 3 animals were found as approximately: liver, 70ng/g, jejunum 14 ng/g, serum 14 ng/g.

At a dose of 100 mg/kg body weight, AL-DUP 5546 was found to leave thelevels of ApoB mRNA present in samples of liver tissue from treated miceessentially unchanged at 95±9% of the mRNA levels present in livertissue of animals receiving carrier only, and levels of ApoB mRNA injejunum were at 102±16% of the levels in control animals, as determinedby the branched-DNA assay. Serum ApoB protein concentration in mousesera was left essentially unchanged at 113±39% of carrier controllevels. Serum cholesterol was elevated to 132±10% of carrier controllevels. Average iRNA concentrations for 3 animals were found asapproximately: liver, 1 ng/g; jejunum, not detectable; serum, notdetectable.

At a dose of 100 mg/kg body weight, AL-DUP 5536 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 56±8% of the mRNA levels present in liver tissue of animals receivingcarrier only, and levels of ApoB mRNA in jejunum were lowered to 28±8%of the levels in control animals, as determined by the branched-DNAassay. Serum ApoB protein concentration in mouse sera was lowered to46±6% of carrier control levels. Serum cholesterol was lowered to 74±33%of carrier control levels. Average iRNA concentrations for 3 animalswere found as approximately: liver, 2 ng/g; jejunum, 6 ng/g, serum, 6ng/g.

At a dose of 100 mg/kg body weight, AL-DUP 5537 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 72±11% of the mRNA levels present in liver tissue of animalsreceiving carrier only, and levels of ApoB mRNA in jejunum wereessentially unchanged at 94±9% of the levels in control animals, asdetermined by the branched-DNA assay. Serum ApoB protein concentrationin mouse sera was left essentially unchanged at 75±25% of carriercontrol levels. Serum cholesterol was left essentially unchanged at118±9% of carrier control levels. Average iRNA concentrations for 3animals were found as approximately: liver, not detectable; jejunum, notdetectable, serum, 1 ng/g.

At a dose of 100 mg/kg body weight, AL-DUP 5538 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 56±16% of the mRNA levels present in liver tissue of animalsreceiving carrier only, and levels of ApoB mRNA in jejunum were loweredto 75±1% of the levels in control animals, as determined by thebranched-DNA assay. Serum ApoB protein concentration in mouse sera wasleft essentially unchanged at 102±27% of carrier control levels. Serumcholesterol left essentially unchanged at 117±18% of carrier controllevels. Average iRNA concentrations for 3 animals were found asapproximately: liver, 35 ng/g; jejunum, 7 ng/g, serum, 18 ng/g.

At a dose of 100 mg/kg body weight, AL-DUP 5539 was found to leave thelevels of ApoB mRNA present in samples of liver tissue from treated miceessentially unchanged at 76±18% of the mRNA levels present in livertissue of animals receiving carrier only, and levels of ApoB mRNA injejunum were lowered to 62±12% of the levels in control animals, asdetermined by the branched-DNA assay. Serum ApoB protein concentrationin mouse sera was left essentially unchanged at 108±23% of carriercontrol levels. Serum cholesterol was left essentially unchanged at102±18% of carrier control levels. Average iRNA concentrations for 3animals were found as approximately: liver, 7 ng/g; jejunum, 4 ng/g,serum, 2 ng/g.

At a dose of 100 mg/kg body weight, AL-DUP 5540 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 54±12% of the mRNA levels present in liver tissue of animalsreceiving carrier only, and levels of ApoB mRNA in jejunum were loweredto 54±12% of the levels in control animals, as determined by thebranched-DNA assay. Serum ApoB protein concentration in mouse sera wasleft essentially unchanged at 72±30% of carrier control levels. Serumcholesterol was left essentially unchanged at 91±10% of carrier controllevels. Average iRNA concentrations for 3 animals were found asapproximately: liver, 130 ng/g; jejunum, 28 ng/g, serum, 25 ng/g.

At a dose of 100 mg/kg body weight, AL-DUP 5541 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 73±10% of the mRNA levels present in liver tissue of animalsreceiving carrier only, and levels of ApoB mRNA in jejunum were loweredto 68±5% of the levels in control animals, as determined by thebranched-DNA assay. Serum ApoB protein concentration in mouse sera wasleft essentially unchanged at 90±8% of carrier control levels. Serumcholesterol was left essentially unchanged at 99±8% of carrier controllevels. Average iRNA concentrations for 3 animals were found asapproximately: liver, 72 ng/g; jejunum, 10 ng/g, serum, 7 ng/g.

At a dose of 100 mg/kg body weight, AL-DUP 5542 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 58±9% of the mRNA levels present in liver tissue of animals receivingcarrier only, and levels of ApoB mRNA in jejunum were lowered to 28±4%of the levels in control animals, as determined by the branched-DNAassay. Serum ApoB protein concentration in mouse sera was lowered to55±9% of carrier control levels. Serum cholesterol was left essentiallyunchanged at 61±27% of carrier control levels. Average iRNAconcentrations for 3 animals were found as approximately: liver, 8 ng/g;jejunum, 17 ng/g, serum, 22 ng/g.

At a dose of 100 mg/kg body weight, AL-DUP 5543 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 77±5% of the mRNA levels present in liver tissue of animals receivingcarrier only, and levels of ApoB mRNA in jejunum were essentiallyunchanged at 91±14% of the levels in control animals, as determined bythe branched-DNA assay. Serum ApoB protein concentration in mouse serawas left essentially unchanged at 97±16% of carrier control levels.Serum cholesterol was left essentially unchanged at 128±24% of carriercontrol levels. Average iRNA concentrations for 3 animals were found asapproximately: liver, not detectable; jejunum, not detectable, serum,not detectable.

At a dose of 100 mg/kg body weight, AL-DUP 5544 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 63±6% of the mRNA levels present in liver tissue of animals receivingcarrier only, and levels of ApoB mRNA in jejunum were lowered to 20±3%of the levels in control animals, as determined by the branched-DNAassay. Serum ApoB protein concentration in mouse sera was lowered to46±5% of carrier control levels. Serum cholesterol was lowered to 55±5%of carrier control levels. Average iRNA concentrations for 3 animalswere found as approximately: liver, >900 ng/g; jejunum, 60 ng/g, serum,40 ng/g.

At a dose of 100 mg/kg body weight, AL-DUP 5545 was found to lower thelevels of ApoB mRNA present in samples of liver tissue from treated miceto 58±11% of the mRNA levels present in liver tissue of animalsreceiving carrier only, and levels of ApoB mRNA in jejunum were loweredto 37±11% of the levels in control animals, as determined by thebranched-DNA assay. Serum ApoB protein concentration in mouse sera waslowered to 50±6% of carrier control levels. Serum cholesterol was leftessentially unchanged at 75±28% of carrier control levels. Average iRNAconcentrations for 3 animals were found as approximately: liver, 70ng/g; jejunum, 7 ng/g, serum, not detectable.

Example 12 Testing siRNAs for Immunogenic Potential

Recently, several reports have been published that postulated apotential of siRNA agents to illicit a possibly adverse immunogenicresponse (see, for example, Hornung et al., Nature Med 2005,11:263-270). Little is known about the biological consequences of, forexample, a temporary interferon-α (IFN-α) increase in humans potentiallycaused by siRNA. To circumvent unnecessary, hazardous side effects, itis desirable to have a potent antiviral siRNA with little or nodetectable immunostimulatory activity. Albeit a true simulation of theexact processes in humans are not possible, we consider the describedexperiment as appropriate for predicting immunostimulation byoligonucleotides and siRNA.

We tested the immunogenicity of the siRNAs listed in Table 11 bymeasuring the induction of IFN-α in peripheral blood mononuclear cells(PBMC) by siRNAs AL-DUP 5167, AL-DUP 5536, AL-DUP 5537, AL DUP 5538, ALDUP 5539, AL DUP 5542, AL-DUP 5543, AL DUP 5544, and AL DUP 5545. AL-DUP5311 was included as an unrelated sequence control. ODN2216, a stronginducer of IFN-α (Hornung et al., Nature Med 2005, 11:263-270) was usedas a positive control, PBS as negatice control. The nucleotide sequenceof ODN2216 is

SEQ. ID NO. 306 5′-

GGGGACGATCGTCGGGGGG-3′

PBMC were isolated by Ficoll gradient centrifugation as described inChang, H. S., and Sack, D. A., Clin. Diag. Lab. Immunology 2001, 8:482-488, except that an unfiltered, erythrocyte depleted leukocyteconcentrate (Buffy Coat) from single donors obtained from the Institutefor Transfusion Medicine gGmbH, Suhl, Germany, diluted 1:1 with PBS, wasemployed as starting material, and that the final suspension in RPMIcomplete medium (RPMI1640 complete; 10% FCS; 1% L-Glu) was adjusted to1×10⁶ cells/ml.

Cells were incubated with ODN2216 or siRNAs in Opti-MEM or Opti-MEM plusthe transfection reagent GenPorter 2 (GP2; Peqlab Biotechnologie GmbH,Erlangen, Germany). 100 μl cell suspension (100.000 cells) per well of a96 well plate were combined with 50 μl of a 1.5 μM solution ofoligonucleotide in Opti-MEM (final oligonucleotide conc. 500 nM), or 50μl of a 1:1 mixture of a) a mixture of 6 μl of GP2 reagent with 119 μlOpti-MEM, and b) a mixture of 1 μl 100 μM solution of oligonucleotide inPBS and 124 μl Diluent B from the GP2 kit (final oligonucleotide conc.133 nM). The incubation was kept at 37° C. for 24 h, and 50 μlsupernatant were carefully removed from the top of the well. These wereemployed for IFN-α determination using the huIFN-α instant ELISA(BenderMed Systems, Vienna, Austria, catalogue no. BMS2161NST). Table 14summarizes the results.

TABLE 14 IFN-α production by peripheral blood mononuclear cellsincubated with siRNAs or ODN2216 Duplex descriptor IFN-α [pg/mlsupernatant] Saline  0 ± 3 ODN 2216 383 ± 62 AL-DUP 5311 77 ± 4 AL-DUP5167 193 ± 9  AL-DUP 5546 159 ± 33 AL-DUP 5536  0 ± 1 AL-DUP 5537  2 ± 1AL-DUP 5538 −5 ± 0 AL-DUP 5539 −10 ± 1  AL-DUP 5542 −3 ± 1 AL-DUP 5543−2 ± 0 AL-DUP 5544 −2 ± 0 AL-DUP 5545 −10 ± 0 

Conclusions from Examples 11 and 12:

a) Oligonucleotides with modified nucleotides in certain particularlydegradation-prone sites benefit in terms of in vitro half life inbiological media while their in vitro and in vivo gene expressioninhibiting activity is largely unaffected

b) Depending on their sequence, siRNAs can be, but are not generally,immunostimulatory agents.

c) AL-DUP 5536, AL-DUP 5540 and AL-DUP 5542 are particularly promisingcandidates as iRNA agents for the inhibition of apoB expression, andtherefore as therapeutics for disorders involving aberrant expression ofapoB.

Table 15 lists the agent numbers that may be used herein to designatethe iRNA agents described above:

TABLE 15 IFN-α production by peripheral blood mononuclear cellsincubated with siRNAs or ODN2216 Duplex descriptor Agent number AL-DUP5163 54 AL-DUP 5164 55 AL-DUP 5165 56 AL-DUP 5166 57 AL-DUP 5167 58AL-DUP 5168 59 AL-DUP 5169 60 AL-DUP 5170 61 AL-DUP 5180 62 AL-DUP 518163 AL-DUP 5182 64 AL-DUP 5183 65 AL-DUP 5536 66 AL-DUP 5537 67 AL-DUP5538 68 AL-DUP 5539 69 AL-DUP 5542 70 AL-DUP 5543 71 AL-DUP 5544 72AL-DUP 5545 73 AL-DUP 5545 74

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

What is claimed is:
 1. An iRNA agent comprising a sense strand and anantisense strand, wherein said sense strand comprises a first sequenceand said antisense strand comprises a second sequence, wherein saidfirst sequence is complementary to said second sequence, wherein saidsecond sequence is complementary to a segment of an Apolipoprotein B(ApoB) mRNA, wherein each strand of said iRNA agent is between 15 and 30nucleotides in length, and wherein the second sequence comprises atleast 15 contiguous nucleotides of the nucleotide sequence of SEQ IDNO:154.
 2. The iRNA agent of claim 1, further comprising anon-nucleotide moiety.
 3. The iRNA agent of claim 1, further comprisinga phosphorothioate linkage.
 4. The iRNA agent of claim 3, wherein saidiRNA agent comprises a phosphorothioate at the first and secondinternucleotide linkage at the 3′ end of the antisense strand.
 5. TheiRNA agent of claim 3, wherein said iRNA agent comprises aphosphorothioate at the first internucleotide linkage at the 3′ end ofthe sense strand.
 6. The iRNA agent of claim 1, further comprising a2′-modified nucleotide.
 7. The iRNA agent of claim 6, wherein the2′-modification is chosen from the group of: 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-0-aminopropyl (2′-O-AP), 2′-0-dimethylaminoethyl (2′-O-DMAOE),2′-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), and 2′-O-N-methylacetamido (2′-O-NMA).
 8. The iRNA agentof claim 6, further comprising: at least one 5′-uridine-adenine-3′(5′-UA-3′) dinucleotide wherein the uridine is a 2′-modified nucleotide;at least one 5′-uridine-guanine-3′ (5′-UG-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide; at least one5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the 5′-cytidineis a 2′-modified nucleotide; or at least one 5′-uridine-uridine-3′(5′-UU-3′) dinucleotide, wherein the 5′-uridine is a 2′-modifiednucleotide.
 9. The iRNA agent of claim 8, wherein the 5′-mostpyrimidines in all occurrences of sequence motif 5′-UA-3′, 5′-CA-3′,5′-UU-3′, and 5′-UG-3′ on the antisense strand are 2′-modifiednucleotides.
 10. The iRNA agent of claim 1, wherein the first sequencecomprises at least 15 contiguous nucleotides of the nucleotide sequenceof SEQ ID NO:153.
 11. The iRNA agent of claim 1, wherein the secondsequence comprises the nucleotide sequence of SEQ ID NO:154.
 12. TheiRNA agent of claim 1, wherein a 5′-end of said iRNA agent comprises anadenosine, cytidine, guanosine, thymidine, or uridine.
 13. The iRNAagent of claim 1, further comprising a nucleotide overhang having 1 to 4nucleotides.
 14. The iRNA agent of claim 13, wherein the nucleotideoverhang has 2 or 3 unpaired nucleotides.
 15. The iRNA agent of claim13, wherein the nucleotide overhang is at the 3′-end of the antisensestrand of the iRNA agent.
 16. The iRNA agent of claim 1, furthercomprising a cholesterol moiety.
 17. The iRNA agent of claim 16, whereinthe cholesterol moiety is conjugated to the 3′-end of the sense strandof the iRNA agent.
 18. The iRNA agent of claim 1, wherein said iRNAagent reduces the amount of ApoB mRNA present in cultured mouse cells ofhepatic origin by an amount greater than a control iRNA agent.
 19. TheiRNA agent of claim 1, wherein the agent reduces the amount of ApoB mRNAin cultured human HepG2 cells after incubation with the agent by morethan 50% compared to cells that have not been incubated with the agent,or reduces the amount of ApoB protein secreted into cell culturesupernatant by more than 50% compared to a control.
 20. A method forreducing the expression levels of ApoB in a subject, comprisingadministering the iRNA agent of claim 1 to said subject.
 21. The iRNAagent of claim 1, wherein the first sequence comprises the nucleotidesequence of SEQ ID NO:153.
 22. The iRNA agent of claim 1, wherein thefirst sequence comprises the nucleotide sequence of SEQ ID NO:153 andthe second sequence comprises the nucleotide sequence of SEQ ID NO:154.